Fadwa Jroundi

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.

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.

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.

Consolidation of archaeological gypsum plaster by bacterial biomineralization of calcium carbonate

Gypsum plasterworks and decorative surfaces are easily degraded, especially when exposed to humidity, and thus they require protection and/or consolidation. However, the conservation of historical gypsum-based structural and decorative materials by conventional organic and inorganic consolidants shows limited efficacy. Here, a new method based on the bioconsolidation capacity of carbonatogenic bacteria inhabiting the material was assayed on historical gypsum plasters and compared with conventional consolidation treatments (ethyl silicate; methylacrylate-ethylmethacrylate copolymer and polyvinyl butyral). Conventional products do not reach in-depth consolidation, typically forming a thin impervious surface layer which blocks pores. In contrast, the bacterial treatment produces vaterite (CaCO3) biocement, which does not block pores and produces a good level of consolidation, both at the surface and in-depth, as shown by drilling resistance measurement system analyses. Transmission electron microscopy analyses show that bacterial vaterite cement formed via oriented aggregation of CaCO3 nanoparticles (∼20nm in size), resulting in mesocrystals which incorporate bacterial biopolymers. Such a biocomposite has superior mechanical properties, thus explaining the fact that drilling resistance of bioconsolidated gypsum plasters is within the range of inorganic calcite materials of equivalent porosity, despite the fact that the bacterial vaterite cement accounts for only a 0.02 solid volume fraction. Bacterial bioconsolidation is proposed for the effective consolidation of this type of material. The potential applications of bacterial calcium carbonate consolidation of gypsum biomaterials used as bone graft substitutes are discussed.

Stone-isolated carbonatogenic bacteria as inoculants in bioconsolidation treatments for historical limestone.

Stone consolidation treatments that use bacterial biomineralization are mainly based on two strategies: (1) the inoculation of a bacterial culture with proven carbonatogenic ability and/or (2) the application of a culture medium capable of activating those bacteria able to induce the formation of calcium carbonate, from amongst the bacterial community of the stone. While the second strategy has been demonstrated to be effective and, unlike first strategy, it does not introduce any exogenous microorganism into the stone, problems may arise when the bacterial community of the stone is altered, for instance by the use of biocides in the cleaning process. In this study we isolate bacteria that belong to the natural microbial community of the stone and which have proven biomineralization capabilities, with the aim of preparing an inoculum that may be used in stone consolidation treatments wherein the natural community of those stones is altered. With this aim, outdoor experiments were undertaken to activate and isolate bacteria that display high biomineralization capacity from altered calcarenite stone. Most of the bacteria precipitated calcium carbonate in the form of calcite. The selected bacteria were phylogenetically affiliated with members of Actinobacteria, Gamma-proteobacteria and Firmicutes. Furthermore, the capability of these selected carbonatogenic bacteria to consolidate altered calcarenite stone slabs was studied in in vitro experiments, both in the presence and the absence of Myxococcus xanthus, as a potential reinforcement for the bacterial biomineralization. Herein, Acinetobacter species, belonging to the microbial community of the stone, are proposed as powerful carbonatogenic bacteria that, inoculated under appropriate conditions, may be used as inoculum for calcareous stone conservation/consolidation in restoration interventions where the microbial community of the stone is altered.

Protection and consolidation of stone heritage by self-inoculation with indigenous carbonatogenic bacterial communities

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.Salt weathering enhanced by global warming and environmental pollution is increasingly threatening stone monuments and artworks. Here, the authors present a bacterial self-inoculation approach with indigenous carbonatogenic bacteria and find that this technique consolidates and protects salt damaged stone.

Role of bacteria in marine barite precipitation : A case study using Mediterranean seawater

Marine bacteria isolated from natural seawater were used to test their capacity to promote barite precipitation under laboratory conditions. Seawater samples were collected in the western and eastern Mediterranean at 250 m and 200 m depths, respectively, since marine barite formation is thought to occur in the upper water column. The results indicate that Pseudoalteromonas sp., Idiomarina sp. and Alteromonas sp. actually precipitate barite under experimental conditions. Barite precipitates show typical characteristics of microbial precipitation in terms of size, morphology and composition. Initially, a P-rich phase precipitates and subsequently evolves to barite crystals with low P contents. Under laboratory conditions barite formation correlates with extracellular polymeric substances (EPS) production. Barite precipitates are particularly abundant in cultures where EPS production is similarly abundant. Our results further support the idea that bacteria may provide appropriate microenvironments for mineral precipitation in the water column. Therefore, bacterial production in the past ocean should be considered when using Ba proxies for paleoproductivity reconstructions.

Myxococcus xanthus Colony Calcification: An Study to Better Understand the Processes Involved in the Formation of this Stromatolite-Like Structure

Calcium carbonate precipitation is a common phenomenon in nature and has been observed to be mediated by a number of microorganisms (for a review, see Castanier et al. 2000; Wright and Oren 2005). Bacterially induced carbonate mineralization is important in a wide range of processes including atmospheric CO2 budgeting (Braissant et al. 2002; Ehrlich 2002), carbonate sediment and rock formation (Riding 2000; Ben Chekroun et al.

Molecular Tools for Monitoring the Ecological Sustainability of a Stone Bio-Consolidation Treatment at the Royal Chapel, Granada

Background Biomineralization processes have recently been applied in situ to protect and consolidate decayed ornamental stone of the Royal Chapel in Granada (Spain). While this promising method has demonstrated its efficacy regarding strengthening of the stone, little is known about its ecological sustainability. Methodology/Principal Findings Here, we report molecular monitoring of the stone-autochthonous microbiota before and at 5, 12 and 30 months after the bio-consolidation treatment (medium/long-term monitoring), employing the well-known molecular strategy of DGGE analyses. Before the bio-consolidation treatment, the bacterial diversity showed the exclusive dominance of Actinobacteria (100%), which decreased in the community (44.2%) after 5 months, and Gamma-proteobacteria (30.24%) and Chloroflexi (25.56%) appeared. After 12 months, Gamma-proteobacteria vanished from the community and Cyanobacteria (22.1%) appeared and remained dominant after thirty months, when the microbiota consisted of Actinobacteria (42.2%) and Cyanobacteria (57.8%) only. Fungal diversity showed that the Ascomycota phylum was dominant before treatment (100%), while, after five months, Basidiomycota (6.38%) appeared on the stone, and vanished again after twelve months. Thirty months after the treatment, the fungal population started to stabilize and Ascomycota dominated on the stone (83.33%) once again. Members of green algae (Chlorophyta, Viridiplantae) appeared on the stone at 5, 12 and 30 months after the treatment and accounted for 4.25%, 84.77% and 16.77%, respectively. Conclusions The results clearly show that, although a temporary shift in the bacterial and fungal diversity was observed during the first five months, most probably promoted by the application of the bio-consolidation treatment, the microbiota tends to regain its initial stability in a few months. Thus, the treatment does not seem to have any negative side effects on the stone-autochthonous microbiota over that time. The molecular strategy employed here is suggested as an efficient monitoring tool to assess the impact on the stone-autochthonous microbiota of the application of biomineralization processes as a restoration/conservation procedure.