María Teresa González-Muñoz

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 CaCO3 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.

Precipitation and Growth Morphology of Calcium Carbonate Induced by Myxococcus Xanthus: Implications for Recognition of Bacterial Carbonates

Abstract It is thought that morphologies of bacterial carbonates can be used to identify microbial fossils and/or precipitates in sediments and rocks. This study shows that calcite and vaterite formed in a gel medium in the presence of Myxococcus xanthus display a range of morphologies that depend on whether the bacteria are live or dead. Metabolic activity of the bacteria induced: (1) aggregates of calcified bacteria formed at maximum supersaturation; (2) vaterite spheres (final growth stage of dumbbell fibrous-radiated aggregates); and (3) dipyramid- and disphenoid-like calcite crystals (combination of {011} and {0001} forms). Morphologies (2) and (3) developed at a lower supersaturation and are typically found in gel-like media. Dipyrimidal-like calcite crystals were also obtained abiotically in gel medium. Dead M. xanthus cells induced heterogeneous precipitation of calcite with rhombohedral morphologies at low supersaturation. A growth mechanism resulting from self-assembly of calcium carbonate nanocrystals may account for the observed morphologies, crystal microstructure, and crystallite size measurements. All of the above-mentioned morphologies of bacterial carbonate have been observed in other laboratory experiments and in continental and marine environments. However, all of them have also been produced abiotically, with the exception of calcified bacterial cells. This may make it more difficult to identify bacterial activity in the rock record. Nonetheless, bacterially induced alkalinization appears to be a prerequisite for the development of spherulitic and dipyramid- or disphenoid-like forms in natural mucilaginous biofilms and microbial mats. The morphologies reported here may facilitate the recognition of early and recent marine and continental microcrystalline bacterial carbonates and cements.

Influence of Substrate Mineralogy on Bacterial Mineralization of Calcium Carbonate: Implications for Stone Conservation

ABSTRACT The 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.

Precipitation of Barite by Myxococcus xanthus: Possible Implications for the Biogeochemical Cycle of Barium

ABSTRACT Bacterial precipitation of barite (BaSO4) under laboratory conditions is reported for the first time. The bacterium Myxococcus xanthus was cultivated in a solid medium with a diluted solution of barium chloride. Crystallization occurred as a result of the presence of live bacteria and the bacterial metabolic activity. A phosphorous-rich amorphous phase preceded the more crystalline barite formation. These experiments may indicate the involvement of bacteria in the barium biogeochemical cycle, which is closely related to the carbon cycle.

Spectroscopic and Microscopic Characterization of Uranium Biomineralization in Myxococcus xanthus

In this work, synchrotron-based X-ray absorption spectroscopy (XAS) and transmission electron microscopy (TEM) studies were carried out to elucidate at molecular scale the interaction mechanisms of Myxococcus xanthus with uranium at different pH values. Extended X-ray absorption fine structure (EXAFS) spectroscopic measurements showed that there are significant differences in the structural parameters of the U complexes formed by this bacterium at pH 2 and 4.5. At very low acidic pH of 2, the cells accumulated U(VI) as organic phosphate-metal complexes. At pH 4.5, however, the cells of this bacterium precipitated U(VI) as meta-autunite-like phase. TEM analyses demonstrated that at pH 2 the uranium accumulates were located mainly at the cell surface, whereas at pH 4.5 a uranium precipitation occurred on the cell wall and within the extracellular polysaccharides (EPS) characteristic of this bacterium. Dead/live staining studies showed that 30% and 50% of the uranium treated cell populations were alive at pH 2 and 4.5, respectively. The precipitation of U(VI) as mineral meta-autunite-like phase is possibly due to the bacterial acidic phosphatase activity. The precipitation of uranium as mineral phases may lead to more stable U(VI) sequestration that may be suitable for remediation purposes. These observations, combined with the very high uranium accumulation capability of the studied bacterial cells indicate that M. xanthus may significantly influence the fate of uranium in soil environments where these bacterial species are mainly found.

Bioconservation of deteriorated monumental calcarenite stone and identification of bacteria with carbonatogenic activity

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.

Bacterial biomineralization: new insights from Myxococcus-induced mineral precipitation

Abstract Bacteria have contributed to the formation of minerals since the advent of life on Earth. Bacterial biomineralization plays a critical role on biogeochemical cycles and has important technological and environmental applications. Despite the numerous efforts to better understand how bacteria induce/mediate or control mineralization, our current knowledge is far from complete. Considering that the number of recent publications on bacterial biomineralization has been overwhelming, here we attempt to show the importance of bacteria–mineral interactions by focusing in a single bacterial genus, Myxococcus, which displays an unusual capacity of producing minerals of varying compositions and morphologies. First, an overview of the recent history of bacterial mineralization, the most common bacteriogenic minerals and current models on bacterial biomineralization is presented. Afterwards a description of myxobacteria is presented, followed by a section where Myxococcus-induced precipitation of a number of phosphates, carbonates, sulphates, chlorides, oxalates and silicates is described and discussed in lieu of the information presented in the first part. As concluding remarks, implications of bacterial mineralization and perspectives for future research are outlined. This review strives to show that the mechanisms which control bacterial biomineralization are not mineral- or bacterial-specific. On the contrary, they appear to be universal and depend on the environment in which bacteria dwell.

Precipitation of barite by marine bacteria: A possible mechanism for marine barite formation

Barite (BaSO 4 ) is found throughout the ocean, yet seawater is undersaturated with respect to barite, and organisms that could account for the abundance of barite have not yet been identifi ed. The mechanism for barite formation in seawater is not fully understood. Here we show that marine bacteria have the ability to precipitate barite through a metabolically mediated biomineralization process. We precipitated barite in laboratory experiments in the presence of several strains of marine bacteria grown on yeast media enriched with barium (Ba); barite did not precipitate under identical conditions in killed-bacteria controls. The crystals develop from amorphous, phosphorous-rich spherical precursors with fi brous internal textures, common in bacterial mineral precipitation. Bacterial mediation of barite precipitation can explain the distribution of barite in the water column and the occurrence of barite crystals in organic-rich sinking aggregates where bacteria are concentrated. This fi nding has implications for the use of barite and Ba proxies in paleoceanographic research.

Molecular monitoring of the microbial dynamics occurring on historical limestone buildings during and after the in situ application of different bio-consolidation treatments

Microbially Induced Carbonate Precipitation is proposed as an environmentally friendly method to protect decayed ornamental stone and introduced in the field of preservation of Cultural Heritage. Recent conservation studies performed under laboratory conditions on non-sterile calcarenite stones have successfully reported on the application of a suitable nutritional solution, inoculated and non-inoculated with Myxococcus xanthus, as a bioconsolidation treatment. Furthermore, this procedure has been applied in situ, very recently, to selected historical buildings in Granada, Spain. For the first time, we evaluate the efficiency and risks of the in situ application of the above mentioned treatments onto two historical buildings in Granada. The evaluation consists of a detailed investigation of the micro-biota actively growing during the seven days of the treatments – short-term monitoring and of that remaining on the stones after six and twelve months of the application – long-term monitoring. A molecular strategy, including DNA extraction, PCR amplification of 16S rRNA sequences, construction of clone libraries and fingerprinting by DGGE (Denaturing Gradient Gel Electrophoresis) analysis followed by sequencing was used to gain insight into the microbial diversity present on the differentially treated stones. The monitoring of M. xanthus was performed by PCR using species-specific primers. Similar dynamics were triggered on both buildings by the application of the nutritional solution (inoculated or non-inoculated). 16S rDNA sequencing revealed the dominant occurrence of members belonging to the Firmicutes and Proteobacteria during the seven days of the treatment, whereas after one year the order Bacillales of the phylum Firmicutes was the predominantly detected microorganisms. M. xanthus could be detected only during the seven days of the treatment. The treatments seem to activate no dangerous microorganisms and furthermore, to select the remainder of a homogeneous group of carbonatogenic bacteria on the stones after a long period of time.

Ca–Mg kutnahorite and struvite production by Idiomarina strains at modern seawater salinities

The production of Mg-rich carbonates by Idiomarina bacteria at modern seawater salinities has been investigated. With this objective, four strains: Idiomarina abyssalis (strain ATCC BAA-312), Idiomarina baltica (strain DSM 15154), Idiomarina loihiensis (strains DSM 15497 and MAH1) were used. The strain I. loihiensis MAH1 is a new isolate, identified in the scope of this work. The four moderately halophilic strains precipitated struvite (NH4MgPO4 x 6H2O) crystals that appear encased by small Ca-Mg kutnahorite [CaMg(CO3)2] spheres and dumbbells, which are also regularly distributed in the bacterial colonies. The proportion of Ca-Mg kutnahorite produced by the bacteria assayed ranged from 50% to 20%, and I. abyssalis also produced monohydrocalcite. All precipitated minerals appeared to be related to the bacterial metabolism and, consequently, can be considered biologically induced. Amino acid metabolism resulted in a release of ammonia and CO2 that increase the pH and CO(3)(2-) concentration of the culture medium, creating an alkaline environment that favoured carbonate and struvite precipitation. This precipitation may be also related to heterogeneous nucleation on negatively charged points of biological structures. Because the nature of the organic matrix determines which ion is preferentially adsorbed and, consequently, which mineral phase is formed, the uniquely high content in odd-iso-branched fatty acids of the Idiomarina suggests that their particular membrane characteristics could induce Ca-Mg kutnahorite production. The Ca-Mg kutnahorite, a mineral with a dolomite-ordered structure, production at seawater salinities is noticeable. To date, such precipitation in laboratory cultures, has only been described in hypersaline conditions. It has also been the first time that biomineralization processes have been related to Idiomarina bacteria.