PRELIMINARY STUDIES ON BIOPRECIPITATION PROCESSES MEDIATED BY SULFATE REDUCING BACTERIA (SRB) AND METAL IMMOBILIZATION IN MINE IMPACTED ENVIRONMENTS.

Mining activity often leaves a critical legacy represented by huge volumes of mine wastes and residues, usually made up of highly reactive materials, which lead to the mobilization and dispersion of harmful elements in soils and waters. Although these extreme environments are adverse to the development of living organisms, it has been observed that some microorganisms are able to adapt, playing a role in metal mobility, and becoming part of the resilience of the system itself. The Iglesiente and Arburese (SW Sardinia, Italy) mine districts, now abandoned, have been exploited for centuries by mining activities aimed at Pb-Zn extraction from sulfides and non-sulfides (calamine) deposits. Here, biogeochemical barriers naturally occur as an adaptation of the ecosystem to environmental stresses. Studies, from macroscale to microscale, showed that sulfate-reducing bacteria (SRB) may influence metal mobility by mediating the precipitation of secondary authigenic metal sulfides under reducing conditions. Specifically, framboids of Zn sulfides and Fe sulfides have been observed in the sections of stream sediments core characterized by the presence of abundant organic matter, especially residues of vegetal tissues (e.g. roots and stems of Juncus acutus and Phragmites australis). Laboratory-scale experiments were performed to better understand the bioprecipitation processes. For this purpose, anaerobic batch tests were carried out using high polluted mining waters (Zn and sulfate concentrations up to 102 and 103 mg/l, respectively) inoculated with native selected sulfate-reducing bacteria from stream sediments collected in the investigated areas. Dramatic decrease (up to 100%) in Zn and sulfate was observed in solutions. Moreover, scanning electron microscopy - energy dispersive spectroscopy (SEM-EDS) analysis, performed on solids recovered at the end of the experiments, showed the presence of precipitates characterized by a tubular morphology and made up by S and Zn. SRB inocula were studied by next-generation sequencing (NGS) approach, with the aim to compare the microbial diversity of the different SRB communities and to search for indigenous novel metal-tolerant sulfidogenic microorganisms. These findings represent a valuable step forward to plan effective bioremediation strategies for reducing metal mobility and dispersion. Also, bioprecipitation mediated by SRB can have great potentialities for metal recovery and our results can help to develop biomining techniques. The authors acknowledge CESA (E58C16000080003) from RAS and RAS/FBS (F72F16003080002) grants, and the CeSAR (Centro Servizi d'Ateneo per la Ricerca) of the University of Cagliari, Italy, for SEM analysis

Sustainable Restoration Through Biotechnological Processes: A Proof of Concept

AbstractAn understanding of the different microbial constellations or microbiomes, which every habitat and every organism harbor, will be the key to addressing many of the challenges humanity will face in the twenty-first century. Such comprehension could launch several innovations relating to natural and cultural capital, including historical and artistic heritage. In relation to cultural heritage, microorganisms are mainly known through their role as deteriogens, but the features creating damage can be exploited positively, attaining more sustainable restoration strategies, in accordance with the principles of compatibility and retreatability deriving from reflections on the Cultural Heritage inspired by the Charter of Venice (International charter for the Conservation and restoration of monument and sites (the Venice Chart 1964). In: ICOMOS, IInd International Congress of Architects and Technicians of Historic Monuments, 1964) onwards. In this article, we show a series of case studies, using both wild-type microorganisms and plant-based extracts, providing a comprehensive proof of concept of the feasibility of biotechnological solutions for a more sustainable restoration strategy, to replace the products in use which are often dangerous for operators, aggressive for works of art and no longer compatible with the environment. The overview of the case studies presented, many of which are still unpublished, responds to the need to go beyond the state of the art and has entirely sprung from suggestions by restorers, interested in learning about potential innovations and strongly determined to introduce non-toxic products in their daily work. In this perspective, the case studies dealt with two topics: bio-cleaning and disinfection. Noteworthy results were obtained on a platform of different types of artworks and different materials with compatible, harmless and selective products

PLANT PRODUCTS AS A GREEN SOLUTION IN THE FIGHT AGAINST BIODETERIORATION OF STONE MONUMENTS

Stone materials in archaeological sites and confined environments are exposed to biodeterioration due to the presence of living organisms that endanger their durability. Weathering and natural or artificial light favour the growth of phototrophic biofilms that are generally composed of cyanobacteria, bacteria, microalgae, mosses and fungi [1]. These microbial communities can cause aesthetic damage to valuable surfaces by causing discoloration and, in some cases, structural problems due to the ability of microorganisms to attack stone, by penetrating the material also through the painted layers. Nowadays, the restoration practices in use include the mechanical removal by brushes combined with spraying chemical biocides that are frequently hazardous towards human health and environment and the effect on the treated surface is not always well understood. Moreover, the employment of chemicals seems to be selective for resistant microorganisms [2]. As a result of these negative factors, more sustainable and safer practices are being investigated for their biocidal action on typical biofilms of lithic surfaces [3-5]. The site of interest for this study is the Domus Aurea (Rome, Italy), built after the fire of 64 AD, as the new residence of the Emperor Nero, the Domus’s walls were covered with precious marbles and vaults decorated with gold and precious stones, then Nero's successors buried the residence, in-filling it entirely with soil. To date, among the restoration efforts to recover the monument, the disinfection of biodeteriogens growing on the walls and ceilings has become a highly important aspect. Based on prior knowledge it was decided to carry out tests on biofilms using the essential oil from Lavandula angustifolia and alcohol extracts from leaves of Glycyrrhizza glabra (Trifolio-M GmbH) and Capsicum spp. These natural compounds were tested at different concentrations, as alternative biocides of phototrophic biofilms. Biofilms samples were collected in ‘room 93’ from an undecorated wall and were then inoculated in agarized BG11 growth medium, to reconstruct homogeneous biofilms to be tested in laboratory. Observations of the biofilms showed that Scytonema julianum was the dominant species. This cyanobacterium has often described from hypogea environments, such as the Roman Catacombs, and it is well known for its biodeteriogenic activity due to the precipitation of calcium carbonate in its sheaths after the dissolution of minerals from the substrate. The tested products were applied to the biofilms twice (day 1 and 5), and the photosynthetic response on the biofilm was followed for five days with a mini-PAM portable fluorometer. As photosynthesis is highly susceptible to this kind of treatment, measurements of rates were used as a proxy for cell health. Changes in photosynthetic activity of the samples treated with the extracts were compared to control biofilms receiving no treatment. Results show that the essential oil of L. angustifolia and G. glabra extract 30% had a higher inhibition potential, followed by G. glabra extract 10%. Capsicum extract was the least efficient. These first results are encouraging enough to continue beyond the laboratory trials in the hope to realise an eco-friendly, non-toxic and sustainable strategy for the conservation of lithic cultural heritage

Killing them gently; control of phototrophic biofilms growing on stone monuments using plant products.

Although lithic archaeological sites and confined environments are relatively harsh environments for microorganisms to inhabit, they are still subjected to biodeterioration by microorganisms able to attack rocks and give rise to a microbial succession (cyanobacteria, bacteria, microalgae, mosses and fungi). These organisms can survive and even grow on minimal amounts of light, water and nutrients, mainly supplied by the substrate and unwitting visitors. Both structural and aesthetic damage to these valuable surfaces occur as a result of physical and chemical processes governed by the biofilm, which result in discoloration and material impairment. Standard physical and chemical treatments to remove biofilms can themselves cause surface damage as well as being health hazard. Moreover, the employment of chemical biocides seems to be selective for resistant microorganisms. Therefore, more sustainable and safer practices are being sought. Rome, 64 AD, Emperor Nero ordered the reconstruction of his residence, with the Domus Aurea forming part of the project to transform Rome into a new city. The main part of the building was located on the Palatine and Oppian Hills and was famous for its sumptuous decoration in which gold and precious stone coverings were added to the stuccos, paintings and coloured marbles, while mosaics, normally restricted to floors were also placed in some of the vaulted ceilings. Nero’s successors were embarrassed of the Domus’ opulence and so stripped it of its marble, its jewels and its ivory within a decade. Within 40 years, the Domus Aurea was completely obliterated and filled in with soil. Surprisingly, this in-filling ensured the survival of the wall paintings by protecting them from damp. After 20 years of excavation and restoration it was opened to the public in 1999, only to close six years later due to detachments and security problems. The doors opened again to the tourist in February of 2007 with restricted visitation. However, due to artificial lighting and high humidity levels many surfaces have been heavily infested by both heterotrophic and phototrophic biofilms. So now, among the main restoration efforts, the disinfection of biodeteriogens growing on the walls and ceilings has become fundamental. Data on the biocidal efficacy of some plant-based products is showing great promise, so it was decided to carry out tests on biofilms sampled at the Domus Aurea using the essential oil from Lavandula angustifolia, alcohol extracts from Glycyrrhizza glabra leaves (Trifolio-M GmbH) and Capsicum sp, singularly or mixed. Biofilm samples were collected in ‘room 93’ from an undecorated wall and were then homogenised and inoculated on agarized BG11 growth medium. Observations showed that the cyanobacterium Scytonema julianum was the dominant species, and has often been described from other hypogean environments, such as Roman Catacombs, and known to deteriorate substrate integrity by dissolution of minerals from the substrate and the precipitation of calcium carbonate on its sheaths. Identification of bacteria by r-DNA16S sequencing revealed the presence of Proteobacteria (6 spp.), Actinobacteria (2 spp.) and Bacteroidetes (1 sp.). Three fungal strains were also isolated and are to be identified. The biofilms were treated twice with the extracts on day 1 and 5, and the photosynthetic response of the biofilm was followed for five days with a mini-PAM portable fluorometer. Photosynthesis is highly susceptible to this kind of treatment, so measurements of rates were used as a proxy for cell health. Changes in photosynthetic activity of the samples treated with the extracts were compared to control biofilms receiving no treatment. Results showed that the essential oil of L. angustifolia and G. glabra leaves extract at 30% had the highest photosynthesis inhibition potential, followed by G. glabra extract 10%. Capsicum extract was the least efficient. These first results are encouraging enough to continue beyond the laboratory trials in the hope to realise an eco-friendly, non-toxic and sustainable strategy for the conservation of lithic cultural heritage

Sustainable Restoration Guided by Scientific and Archival Investigations: The Bio-Cleaning of Lorenzo Duke of Urbino’s Sarcophagus, a Michelangelo’s Masterpiece in the Medici Chapels

The masterpiece this work deals with is the tomb of Lorenzo de’ Medici, Duke of Urbino, completed by Michelangelo around 1533 in the New Sacristy of Medici chapel in Florence. Sacristy underwent an extensive restoration (2013–2020) and Lorenzo’s sarcophagus was addressed in the last phase (2019–2020). The unique history and the mediocre state of conservation of the marble may be related to body decomposition processes that, according to the documentary research, required a scientific approach to tailor an appropriate intervention. The sarcophagus underwent a bio-cleaning procedure, using bacteria belonging to the ENEA-MIRRI (Microbial Resource Research Infrastructure Italian Node) collection. Spectroscopic analysis (FTIR in ATR and in TR—total reflection—mode and XRF) and microscopic analyses allowed a correct diagnosis of the composition of the coherent deposits, guiding the selection of appropriate bacterial strains. Deposits were composed of gypsum, calcium oxalate, calcium phosphates and proteins. Following a laboratory screening and a preliminary test on-site, the three strains, Serratia ficaria SH7, Pseudomonas stutzeri CONC11 and Rhodococcus sp. ZCONT, immobilised in an appropriate supportant gel, were selected by the quality of cleaning obtained. Two applications of the micro-packs, containing each individual strain, were able to remove the centuries-old deposits within 48 h. The bio-cleaning was selective, gradual and respectful of the marble, in accordance with the restorers’ opinion and reflecting the principles of sustainability

Microscopic processes ruling the bioavailability of Zn to roots of Euphorbia pithyusa L. pioneer plant

Euphorbia pithyusa L. was used in a plant growth-promoting assisted field trial experiment. To unravel the microscopic processes at the interface, thin slices of E. pithyusa roots were investigated by micro-X-ray fluorescence mapping. Roots and rhizosphere materials were examined by X-ray absorption spectroscopy at the Zn K-edge, X-ray diffraction, and scanning electron microscopy. Results indicate some features common to all the investigated samples. (i) In the rhizosphere of E. pithyusa, Zn was found to exist in different phases. (ii) Si and Al are mainly concentrated in a rim at the epidermis of the roots. (iii) Zn is mostly stored in root epidermis and does not appear to be coordinated to organic molecules but mainly occurs in mineral phases such as Zn silicates. We interpreted that roots of E. pithyusa significantly promote mineral evolution in the rhizosphere. Concomitantly, the plant uses Si and Al extracted by soil minerals to build a biomineralization rim, which can capture Zn. This Zn silicate biomineralization has relevant implications for phytoremediation techniques and for further biotechnology development, which can be better designed and developed after specific knowledge of molecular processes ruling mineral evolution and biomineralization processes has been gained

Assessment of the applicability of a "toolbox" designed for microbially assisted phytoremediation : the case study at Ingurtosu mining site (Italy)

The paper describes the fieldwork at the Italian test site of the abandoned mine of sphalerite and galena in Ingurtosu (Sardinia), with the aim to assess the applicability of a "toolbox" to establish the optimized techniques for remediation of soils contaminated by mining activities. A preliminary characterization-including (hydro)geochemistry, heavy metal concentration and their mobility in soil, bioprospecting for microbiology and botany-provided a data set for the development of a toolbox to deliver a microbially assisted phytoremediation process. Euphorbia pithyusa was selected as an endemic pioneer plant to be associated with a bacterial consortium, established with ten selected native strains, including metal-tolerant bacteria and producers of plant growth factors. The toolbox was firstly assessed in a greenhouse pot experiment. A positive effect of bacterial inoculum on E. pithyusa germination and total plant survival was observed. E. pithyusa showed to be a well-performing metallophyte species, and only inoculated soil retained a microbial activity with a high functional diversity, expanding metabolic affinity also towards root exudates. These results supported the decision to proceed with a field trial, investigating different treatments used singly or in combination: bioaugmentation with bacterial consortia, mycorrhizal fungi and a commercial mineral amendment. Microbial activity in soil, plant physiological parameters and heavy metal content in plants and in soil were monitored. Five months after the beginning, an early assessment of the toolbox under field conditions was carried out. Despite the cold season (October-March), results suggested the following: (1) the field setup as well as the experimental design proved to be effective; (2) plant survival was satisfactory; (3) soil quality was increased and bioaugmentation improved microbial activity, expanding the metabolic competences towards plant interaction (root exudates); and (4) multivariate analysis supported the data provided that the proposed toolbox can be established and the field trial can be carried forwar