Microbes for Archaeological Wood Conservation.

This project focuses on innovative biological methods of extraction for the preservation of waterlogged wood suffering from salt precipitation and acidification. The principal investigator and her team proposed to exploit biomineralization capacities of some bacteria for anticipating the extraction of iron and sulfur compounds when wood is still wet. A comprehensive assessment of the extraction performances achieved on wood objects from lake and marine environments will allow a versatile extraction method to be proposed to end-users.

Evaluation of an alternative biotreatment for the extraction of harmful iron and sulfur species from waterlogged wood.

An innovative bioextraction method was tested and compared to common chemical extraction for the preservation of waterlogged archeological wood (WAW) artifacts. During burial, WAW artifacts accumulate iron and sulfur species forming iron sulfides. These compounds are harmless in the burial environment, where the oxygen content is low. But upon excavation, the WAW undergoes the oxidation of these compounds, and thus, irreversible physical and chemical damages occur. Fresh and archeological oak and pine samples were selected as representative species of WAW artifacts. Fresh samples were previously artificially contaminated to ascertain the presence of iron and sulfur. Thiobacillus denitrificans and natural iron chelators, called siderophores, were investigated to extract iron and sulfur as a 2-step biological treatment (BT) and compared to sodium persulfate-EDTA as chemical treatment (CT). Consolidation and freeze-drying were performed on the samples after BT and CT as traditional conservation protocols. BT and CT efficiency was evaluated through Raman, inductively coupled plasma-optical emission (ICP-OES), and Fourier transformed infrared (FTIR) spectroscopies. Raman and ICP showed that most of the iron and sulfur was extracted after BT, while some sulfur species remained present on CT samples. None of the extraction methods resulted in a degradation of the wood, as ascertained by FTIR analyses. Yet, all samples presented visual modifications after conservation. Pine samples treated with BT illustrated the oxidation of the species. Present principal component analysis (PCA) and analysis of variance (ANOVA) which were selected as statistical approaches and validated BT as a promising alternative extraction method, with encouraging extraction rates and less alteration of the sample appearance.

Biological oxidation of iron sulfides.

The biological oxidation of minerals and ores, called bioleaching, has been studied for the last decades to solubilize metals and recover them. In particular, iron sulfides are the most studied ores for an optimum extraction of different metals, such as copper or zinc. The use of chemolithotrophic bacteria, as Acidothiobacillus ferrooxidans, to oxidize both iron and sulfur species in aerobic conditions and at acidic pH shows promising results. In the field of heritage preservation, the development of "green" treatments is more and more studied. Waterlogged archeological wood presents an accumulation of iron sulfides within its structure, which, after exposition to oxygen, lead to salt precipitation and acidification and so to the degradation of the wooden artifact. A new extraction method, based on the dissolution of iron sulfides by the use of bacteria could be an alternative to the current chemical extraction methods, as being more respectful and ecological. While A. ferrooxidans is very effective in mines and groundwater, in the field of conservation-restoration of wood, Thiobacillus denitrificans is a better candidate as it grows at neutral pH, which is less aggressive for organic substrates (wood here). Preliminary studies show the efficiency of T. denitrificans for the dissolution of iron sulfides, as the concentration of nitrates used as electron donors decreases while the concentration of sulfates produced increases without degrading the wooden matrix. Long-term behavior should be studied to assess the stability of the artifacts after treatment.

Siderophores: From natural roles to potential applications.

Siderophores are secondary metabolites produced by different organisms in order to scavenge iron from their surrounding environment making this essential element available to the cell. Presenting high affinity for ferric iron, siderophores are secreted out to form soluble ferric complexes that can be taken up by the organisms. Siderophores present complex chemistry that allows them to form the strongest iron-chelating complexes. Interest in this field is always up to date and new siderophores are found with new roles and applications. For example, siderophores participate to the mobilization of iron and other elements and are involved in virulence processes. Recently, a strong relation between siderophores and oxidative stress tolerance has been also highlighted. Their application in medicine has been widely studied as well as in agriculture. However, new fields are paying attention to the use of siderophores as green-iron chelators. In particular, siderophores have been proposed for the preservation of cultural heritage.

Remedial Treatment of Corroded Iron Objects by Environmental Aeromonas Isolates.

Using bacteria to transform reactive corrosion products into stable compounds represents an alternative to traditional methods employed in iron conservation. Two environmental Aeromonas strains (CA23 and CU5) were used to transform ferric iron corrosion products (goethite and lepidocrocite) into stable ferrous iron-bearing minerals (vivianite and siderite). A genomic and transcriptomic approach was used to analyze the metabolic traits of these strains and to evaluate their pathogenic potential. Although genes involved in solid-phase iron reduction were identified, key genes present in other environmental iron-reducing species are missing from the genome of CU5. Several pathogenicity factors were identified in the genomes of both strains, but none of these was expressed under iron reduction conditions. Additional in vivo tests showed hemolytic and cytotoxic activities for strain CA23 but not for strain CU5. Both strains were easily inactivated using ethanol and heat. Nonetheless, given a lesser potential for a pathogenic lifestyle, CU5 is the most promising candidate for the development of a bio-based iron conservation method stabilizing iron corrosion. Based on all the results, a prototype treatment was established using archaeological items. On those, the conversion of reactive corrosion products and the formation of a homogenous layer of biogenic iron minerals were achieved. This study shows how naturally occurring microorganisms and their metabolic capabilities can be used to develop bio-inspired solutions to the problem of metal corrosion.IMPORTANCE Microbiology can greatly help in the quest for a sustainable solution to the problem of iron corrosion, which causes important economic losses in a wide range of fields, including the protection of cultural heritage and building materials. Using bacteria to transform reactive and unstable corrosion products into more-stable compounds represents a promising approach. The overall aim of this study was to develop a method for the conservation and restoration of corroded iron items, starting from the isolation of iron-reducing bacteria from natural environments. This resulted in the identification of a suitable candidate (Aeromonas sp. strain CU5) that mediates the formation of desirable minerals at the surfaces of the objects. This led to the proof of concept of an application method on real objects.