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.

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.

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.

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.

Use of Bacteria To Stabilize Archaeological Iron.

Iron artifacts are common among the findings of archaeological excavations. The corrosion layer formed on these objects requires stabilization after their recovery, without which the destruction of the item due to physicochemical damage is likely. Current technologies for stabilizing the corrosion layer are lengthy and generate hazardous waste products. Therefore, there is a pressing need for an alternative method for stabilizing the corrosion layer on iron objects. The aim of this study was to evaluate an alternative conservation-restoration method using bacteria. For this, anaerobic iron reduction leading to the formation of stable iron minerals in the presence of chlorine was investigated for two strains of Desulfitobacterium hafniense (strains TCE1 and LBE). Iron reduction was observed for soluble Fe(III) phases as well as for akaganeite, the most troublesome iron compound in the corrosion layer of archaeological iron objects. In terms of biogenic mineral production, differential efficiencies were observed in assays performed on corroded iron coupons. Strain TCE1 produced a homogeneous layer of vivianite covering 80% of the corroded surface, while on the coupons treated with strain LBE, only 10% of the surface was covered by the same mineral. Finally, an attempt to reduce iron on archaeological objects was performed with strain TCE1, which led to the formation of both biogenic vivianite and magnetite on the surface of the artifacts. These results demonstrate the potential of this biological treatment for stabilizing archaeological iron as a promising alternative to traditional conservation-restoration methods.IMPORTANCE Since the Iron Age, iron has been a fundamental material for the building of objects used in everyday life. However, due to its reactivity, iron can be easily corroded, and the physical stability of the object built is at risk. This is particularly true for archaeological objects on which a potentially unstable corrosion layer is formed during the time the object is buried. After excavation, changes in environmental conditions (e.g., higher oxygen concentration or lower humidity) alter the stability of the corrosion layer and can lead to the total destruction of the object. In this study, we demonstrate the feasibility of an innovative treatment based on bacterial iron reduction and biogenic mineral formation to stabilize the corrosion layer and protect these objects.

Fungal Biorecovery of Gold From E-waste.

Waste electric and electronic devices (e-waste) represent a source of valuable raw materials of great interest, and in the case of metals, e-waste might become a prized alternative source. Regarding gold, natural ores are difficult to mine due to their refractory nature and the richest ores have almost all been exploited. Additionally, some gold mining areas are present in geopolitically unstable regions. Finally, the gold mining industry produces toxic compounds, such as cyanides. As a result, the gold present in e-waste represents a nonnegligible resource (urban mining). Extraction methods of gold from natural ores (pyro- and hydrometallurgy) have been adapted to this particular type of matrix. However, to propose novel approaches with a lower environmental footprint, biotechnological methods using microorganisms are being developed (biometallurgy). These processes use the extensive metabolic potential of microbes (algae, bacteria, and fungi) to mobilize and immobilize gold from urban and industrial sources. In this review, we focus on the use of fungi for gold biomining. Fungi interact with gold by mobilizing it through mechanical attack as well as through biochemical leaching by the production of cyanides. Moreover, fungi are also able to release Au through the degradation of cyanide from aurocyanide complexes. Finally, fungi immobilize gold through biosorption, bioaccumulation, and biomineralization, in particular, as gold nanoparticles. Overall, the diversity of mechanisms of gold recycling using fungi combined with their filamentous lifestyle, which allows them to thrive in heterogeneous and solid environments such as e-waste, makes fungi an important bioresource to be harnessed for the biorecovery of gold.

Agar and Chitosan Hydrogels' Design for Metal-Uptaking Treatments.

In the field of cultural heritage, the use of natural gels is rising for the application of active agents. Here, two natural polymers are assessed: agar, a pioneer hydrogel for conservation treatments, and chitosan, a rather novel and metal-binding gel. For chitosan, a state-of-the-art based formulation (CS-ItA-LCys) is evaluated as it was reported for silver-complexing properties. It is evaluated whether these polymers can withstand the addition of the chelating compound deferoxamine, which is a bacterial siderophore. This allows for the obtainment of completely bio-sourced gel systems. A Fourier-transformed (FT) infrared spectroscopy characterization is performed, completed with rheological measurements and Cryo-Scanning Electron Microscopy (cryo-SEM) to investigate the physico-chemical properties of the gels, as well as their interaction with deferoxamine. Both polymers are also tested for their inherent complexing ability on silver ions using FT-Raman spectroscopy. A multi-analytical comparison shows different microstructures, in particular, the presence of a thick membrane for chitosan and different mechanical behaviors, with agar being more brittle. Neither hydrogel seems affected by the addition of deferoxamine; this is shown by similar rheological behavior and molecular structures in the presence or absence of the chelator. The intrinsic abilities of the chitosan formulation to make silver complex are demonstrated with the observation of two peaks characteristic of Ag-S and Ag-O bonds. Agar and chitosan are both proven to be reliable gels to act as carriers for bio-based active agents. This paper confirms the potential asset of the chitosan formulation CS-ItA-LCys as a promising gel for the complexation of soluble silver.

Innovative perspective for the cleaning of historical iron heritage: novel bio-organogel for the combined removal of undesired organic coatings and corrosion.

An innovative green organogel was designed to simultaneously tackle inorganic compounds (i.e., iron corrosion) and organic substances (i.e., acrylic coatings) as undesired materials possibly present on the surface of altered indoor metal artworks. Poly-3-hydroxybutyrate (PHB), ethyl lactate (EL), and deferoxamine B (DFO) were employed in the formulation as thickening agent, organic solvent, and complexing agent, respectively, aiming to propose a sustainable and less harmful chemical cleaning method for metal care. The components were selected because they are bio-sourced, renewable, biodegradable, and non- or low-toxic materials. A multi-modal protocol of analysis was carried out to characterise the newly designed PHB-EL-DFO organogel. The cleaning performance of the novel formulation was assessed on mild steel mock-ups presenting both corrosion and organic coating to be removed. The conducted multi-analytical approach verified that the PHB-EL-DFO gel was able to tackle the two undesired materials simultaneously in an adjustable and easy-to-use way thanks to a modular application. Supplementary Information: The online version contains supplementary material available at 10.1186/s40494-024-01288-0.

Testing of the siderophore deferoxamine amended in hydrogels for the cleaning of iron corrosion.

Bioderived alternatives to commonly used complexing agents for the cleaning of iron artworks are sought for their natural origin and better biodegradability. Indeed, complexing agents currently used for the removal of undesired corrosion products from iron artworks can be difficult to control and their environmental impact is often overlooked. This paper studies the use of siderophores, focusing on the ability of one of them, deferoxamine, to be employed as an active agent loaded in polysaccharides hydrogels, on corrosion phases. Preliminary tests were conducted on artificially aged steel samples and further studies were performed on naturally corroded steel to assess the most performing application parameters. Long-term behavior of cleaned surface was assessed. Cleaning outcomes were compared with those obtainable with disodium ethylenediaminetetraacetic acid using optical microscopy, colorimetry and atomic absorption spectroscopy as well as Infrared and Raman micro-spectroscopies. Among the different gelling agents evaluated, agar applied when hot and gellan gum prepared at room temperature were the most effective gel formulations and agar left few residues over the treated surfaces. The protocol was then tested on altered steel artifacts belonging to heritage institutions in France. Encouraging outcomes in the removal of iron corrosion phases with green approaches are here presented.

Development of an analytical procedure for evaluation of the protective behaviour of innovative fungal patinas on archaeological and artistic metal artefacts.

In the literature, the ability to transform metal compounds into metal oxalates has been reported for different species of fungi. This could be an innovative conservation method for archaeological and artistic metal artefacts. In fact, with a high degree of insolubility and chemical stability even in acid atmospheres (pH 3), metal oxalates provide the surface with good protection. Within the framework of the EU-ARTECH project, different fungal strains have been used to transform existing corrosion patinas on outdoor bronze monuments into copper oxalates, while preserving the physical appearance of these artefacts. Given the promising results obtained with this first attempt, the same approach is now applied within the BAHAMAS (Marie Curie Intra European Fellowship action) project, but extended to other metal substrates, for example iron and silver, which are frequently found in cultural heritage artworks and also encounter several problems of active corrosion. The research is investigating the formation mechanisms and adhesion properties of the newly formed metal oxalates by means of complementary analytical techniques (X-ray diffraction (XRD), FTIR microscopy, Raman microscopy, scanning electron microscopy (SEM-EDS), electrochemical impedance spectroscopy (EIS), colorimetry). For each metal substrate, the most appropriate fungal strain is going to be identified and applied to corroded sheets and the novel fungal treatment compared with those used so far. Treated metal sheets will be monitored during 1-year exposure to different cycles of artificial ageing, to evaluate the corrosion resistance of the fungal patinas obtained. The objective of this contribution is to present the first results achieved so far on naturally corroded bronze sheets during the EU-ARTECH project and the analytical procedure used for the testing of the proposed treatment performances during the BAHAMAS project.

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.

Performance evaluation of mapping and linear imaging FTIR microspectroscopy for the characterisation of paint cross sections.

Different Fourier transform infrared microspectroscopic techniques, using attenuated total reflection (ATR) mode and single-element mercury-cadmium-telluride (MCT) detector (mapping) or multielement MCT detector (raster scanning), are compared with each other for the characterisation of inorganic compounds and organic substances in paint cross sections. All measurements have been performed on paint cross sections embedded in potassium bromide, a transparent salt in the mid-infrared region, in order to better identify the organic materials without the interference of the usual embedding resin. The limitations and advantages of the different techniques are presented in terms of spatial resolution, data quality and chemical information achieved. For all techniques, the chemical information obtained is found to be nearly identical. However, ATR mapping performed with a recently developed instrumentation shows the best results in terms of spectral quality and spatial resolution. In fact, thin organic layers (approximately 10 microm) have been not only identified but also accurately located. This paper also highlights the recent introduction of multielement detectors, which may represent a good compromise between mapping and imaging systems.

Metabolic processes applied to endangered metal and wood heritage objects: Call a microbial plumber!

While often considered as harmful for cultural heritage, microorganisms can also be used for its safeguarding. The methods used so far for the conservation-restoration of cultural heritage are often unsatisfactory in terms of efficiency and durability. Inhibitors or complexing agents are also toxic and pose potential threats to human health and to the environment. Microbial-based technologies can provide sustainable solutions for heritage conservation-restoration using ecologically friendly biological treatments. Over the last decades, the development of biological methods and materials has become a significant alternative for the preservation of ancient heritage. Of particular note, microbial metabolisms are exploited to consolidate, clean, stabilize or even protect surfaces of cultural items. Taking advantage of unique properties of microorganisms, reactive corrosion products are extracted or converted into biogenic minerals that provide the treated surfaces with long-term stability. Examples of the techniques proposed include the formation of passivating biogenic layers that can be applied for preservation of metal-based heritage, as well as the development of methods for the preventative removal of iron species from waterlogged wood. This review presents the current advance made in research aiming to preserve copper- and iron-based artefacts, in particular sculptures but also archaeological objects, as well as in the development of a method for the extraction of iron species from waterlogged wood.

Investigation of Biogenic Passivating Layers on Corroded Iron.

This study evaluates mechanisms of biogenic mineral formation induced by bacterial iron reduction for the stabilization of corroded iron. As an example, the Desulfitobacterium hafniense strain TCE1 was employed to treat corroded coupons presenting urban natural atmospheric corrosion, and spectroscopic investigations were performed on the samples' cross-sections to evaluate the corrosion stratigraphy. The treated samples presented a protective continuous layer of iron phosphates (vivianite Fe2+3(PO4)2·8H2O and barbosalite Fe2+Fe3+2(PO4)2(OH)2), which covered 92% of the surface and was associated with a decrease in the thickness of the original corrosion layer. The results allow us to better understand the conversion of reactive corrosion products into stable biogenic minerals, as well as to identify important criteria for the design of a green alternative treatment for the stabilization of corroded iron.

ATR and transmission analysis of pigments by means of far infrared spectroscopy.

In the field of FTIR spectroscopy, the far infrared (FIR) spectral region has been so far less investigated than the mid-infrared (MIR), even though it presents great advantages in the characterization of those inorganic compounds, which are inactive in the MIR, such as some art pigments, corrosion products, etc. Furthermore, FIR spectroscopy is complementary to Raman spectroscopy if the fluorescence effects caused by the latter analytical technique are considered. In this paper, ATR in the FIR region is proposed as an alternative method to transmission for the analyses of pigments. This methodology was selected in order to reduce the sample amount needed for analysis, which is a must when examining cultural heritage materials. A selection of pigments have been analyzed in both ATR and transmission mode, and the resulting spectra were compared with each other. To better perform this comparison, an evaluation of the possible effect induced by the thermal treatment needed for the preparation of the polyethylene pellets on the transmission spectra of the samples has been carried out. Therefore, pigments have been analyzed in ATR mode before and after heating them at the same temperature employed for the polyethylene pellet preparation. The results showed that while the heating treatment causes only small changes in the intensity of some bands, the ATR spectra were characterized by differences in both intensity and band shifts towards lower frequencies if compared with those recorded in transmission mode. All pigments' transmission and ATR spectra are presented and discussed, and the ATR method was validated on a real case study.

Soluble and solid iron reduction assays with Desulfitobacterium hafniense.

There is a pressing need to develop sustainable and efficient methods to protect and stabilize iron objects. To develop a conservation-restoration method for corroded iron objects, this bio-protocol presents the steps to investigate reductive dissolution of ferric iron and biogenic production of stabilizing ferrous iron minerals in the strict anaerobe Desulfitobacterium hafniense (strains TCE1 and LBE). We investigated iron reduction using three different Fe(III) sources: Fe(III)-citrate (a soluble phase), akaganeite (solid iron phase), and corroded coupons. This protocol describes a method that combines spectrophotometric quantification of the complex Fe(II)-Ferrozine® with mineral characterization by scanning electron microscopy and Raman spectroscopy. These three methods allow assessing reductive dissolution of ferric iron and biogenic mineral production as a promising alternative for the development of an innovative sustainable method for the stabilization of corroded iron.

Bacterial iron reduction and biogenic mineral formation for the stabilisation of corroded iron objects.

Exploiting bacterial metabolism for the stabilisation of corroded iron artefacts is a promising alternative to conventional conservation-restoration methods. Bacterial iron reduction coupled to biogenic mineral formation has been shown to promote the conversion of reactive into stable corrosion products that are integrated into the natural corrosion layer of the object. However, in order to stabilise iron corrosion, the formation of specific biogenic minerals is essential. In this study, we used the facultative anaerobe Shewanella loihica for the production of stable biogenic iron minerals under controlled chemical conditions. The biogenic formation of crystalline iron phosphates was observed after iron reduction in a solution containing Fe(III) citrate. When the same biological treatment was applied on corroded iron plates, a layer composed of iron phosphates and iron carbonates was formed. Surface and cross-section analyses demonstrated that these two stable corrosion products replaced 81% of the reactive corrosion layer after two weeks of treatment. Such results demonstrate the potential of a biological treatment in the development of a stabilisation method to preserve corroded iron objects.

Microbial biotechnology approaches to mitigating the deterioration of construction and heritage materials.

Microorganisms are the main engines of elemental cycling in this planet and therefore have a profound impact on both organic and mineral substrates. As such, past and present human-made structures and cultural heritage can be negatively affected by microbial activity. Processes such as bioweathering (rocks and minerals), biodeterioration (organic substrates) or biocorrosion (metals) participate to the degradation or structural damage of construction and heritage materials. This structural damage can cause major economic losses (e.g. replacement of cast-iron pipes in water distribution networks), and in the case of heritage materials, the entire loss of invaluable objects or monuments. Even though one can regard the influence of microbial activity on construction and heritage materials as negative, remarkably, the same metabolic pathways involved in degradation can be exploited to increase the stability of these materials.

Evaluation of the performances of a biological treatment on tin-enriched bronze.

Recently, research gives emphasis to eco-friendly and sustainable approaches for the preservation of cultural heritage that could offer advantages in terms of compatibility, durability and safety. Hence, a biological treatment, based on a specific fungal strain of Beauveria bassiana, is exploited for the stabilization of soluble and/or active bronze corrosion products, converting them into copper oxalates. The chemical stability of the latter represents a real improvement for the long-term preservation of bronze, especially in case of exposure to acid rain. However, the corrosion behaviour of bronze differs from that of pure copper due to the presence of additional alloying elements. In natural environments, the selective dissolution of copper leads to a relative tin-enrichment within the corrosion layers, mostly in unsheltered areas exposed to rainwater runoff. To understand the influence of tin-enrichment on the formation of oxalates, pure tin and artificially tin-enriched bronze coupons were treated with this novel biological system and, in the case of bronze coupons, exposed to accelerated ageing. Tin enrichment and accelerated ageing were performed through runoff tests. Before and after treatment and ageing, the sample surface was characterized through Fourier transform infrared (FTIR) and Raman spectroscopies, scanning electron microscopy coupled to energy dispersive spectroscopy (SEM-EDS). Metals released in the ageing solutions were analysed through atomic absorption spectrometry (AAS). The analytical results allowed to better understand the response of unsheltered areas from outdoor bronze monuments to the biological treatment proposed.

Protection of Metal Artifacts with the Formation of Metal-Oxalates Complexes by Beauveria bassiana.

Several fungi present high tolerance to toxic metals and some are able to transform metals into metal-oxalate complexes. In this study, the ability of Beauveria bassiana to produce copper oxalates was evaluated. Growth performance was tested on various copper-containing media. B. bassiana proved highly resistant to copper, tolerating concentrations of up to 20 g L(-1), and precipitating copper oxalates on all media tested. Chromatographic analyses showed that this species produced oxalic acid as sole metal chelator. The production of metal-oxalates can be used in the restoration and conservation of archeological and modern metal artifacts. The production of copper oxalates was confirmed directly using metallic pieces (both archeological and modern). The conversion of corrosion products into copper oxalates was demonstrated as well. In order to assess whether the capability of B. bassiana to produce metal-oxalates could be applied to other metals, iron and silver were tested as well. Iron appears to be directly sequestered in the wall of the fungal hyphae forming oxalates. However, the formation of a homogeneous layer on the object is not yet optimal. On silver, a co-precipitation of copper and silver oxalates occurred. As this greenish patina would not be acceptable on silver objects, silver reduction was explored as a tarnishing remediation. First experiments showed the transformation of silver nitrate into nanoparticles of elemental silver by an unknown extracellular mechanism. The production of copper oxalates is immediately applicable for the conservation of copper-based artifacts. For iron and silver this is not yet the case. However, the vast ability of B. bassiana to transform toxic metals using different immobilization mechanisms seems to offer considerable possibilities for industrial applications, such as the bioremediation of contaminated soils or the green synthesis of chemicals.