Andrea Polo

Feasibility of Removing Surface Deposits on Stone Using Biological and Chemical Remediation Methods

The study was conducted on alterations found on stone artwork and integrates microbial control and a biotechnological method for the removal of undesirable chemical substances. The Demetra and Cronos sculptures are two of 12 stone statues decorating the courtyard of the Buonconsiglio Castle in Trento (Italy). An initial inspection of the statues revealed putative black crusts and highlighted the microbial contamination causing discoloration. In 2006, the Cultural Heritage Superintendence of Trento commissioned us to study and remove these chemical and biological stains. Stereomicroscopy characterised the stone of the sculptures as oolitic limestone, and infrared analyses confirmed the presence of black crusts. To remove the black crusts, we applied a remediation treatment of sulphate-reducing bacteria, which removes the chemical alteration but preserves the original stone and the patina noble. Using traditional and biomolecular methods, we studied the putative microbial contamination and confirmed the presence of biodeteriogens and chose biocide Biotin N for the removal of the agents causing the discolouration. Denaturing gradient gel electrophoresis fluorescent in situ hybridisation established that Cyanobacteria and green algae genera were responsible for the green staining whereas the black microbial contamination was due to dematiaceous fungi. After the biocide Biotin N treatment, we applied molecular methods and demonstrated that the Cyanobacteria, and most of the green algae and dematiaceous fungi, had been efficiently removed. The reported case study reveals that conservators can benefit from an integrated biotechnological approach aimed at the biocleaning of chemical alterations and the abatement of biodeteriogens.

Biofilm Formation in Food Processing Environments is Still Poorly Understood and Controlled

The presence of undesirable biofilms on food processing contact surfaces may lead to: (1) transmission of diseases; (2) food spoilage; (3) shortened time between cleaning events; (4) contamination of product by nonstarter bacteria; (5) metal corrosion in pipelines and tanks; (6) reduced heat transfer efficacy or even obstruction of the heat equipment. Despite the significant problems caused by biofilms in the food industry, biofilm formation in these environments is still poorly understood and effective control of biofilms remains challenging. Although it is understood that cell attachment and biofilm formation are influenced by several factors, including type of strain, chemical–physical properties of the surface, temperature, growth media and the presence of other microorganisms, some conflicting statements can be retrieved from the literature and there are no general trends yet that allow us to easily predict biofilm development. It is likely that still unexplored interaction of factors may be more critical than the effect of a single parameter. New alternative biofilm control strategies, such as biocontrol, use of enzymes and phages and cell-to-cell communication interference, are now available that can reduce the use of chemical agents. In addition, as preventing biofilm formation is a more efficient strategy than controlling mature biofilm, the use of surface-modified materials have been suggested. These strategies may better reveal their beneficial potential when the ecological complexity of biofilms in food environments is addressed.

Effects of Photoactivated Titanium Dioxide Nanopowders and Coating on Planktonic and Biofilm Growth of Pseudomonas aeruginosa

We exploited the ability of photocatalytic titanium dioxide (TiO2) as an agent for the biofilm control. Two photocatalytic systems were investigated: a 3 g L−1 suspension of TiO2 nanopowder in demineralized water and glass slides coated with a TiO2 thin film, achieved by sol‐gel deposition. A running protocol for the photoactivation of TiO2 was set up using the dye rhodamine B. The microorganisms studied were Pseudomonas stutzeri, Pseudomonas aeruginosa and a Bacillus cereus‐group as planktonic cells. P. aeruginosa biofilms were also studied at both the solid‐liquid and the solid‐air interface. The TiO2 nanopowder produced 1‐log reduction of Bacillus sp. planktonic cells in 24 h, 2‐log reduction of P. stutzeri planktonic cells in 30 min and 1‐log reduction of P. aeruginosa planktonic cells in 2 h compared with non–photo‐activated TiO2. TiO2 thin film produced almost a complete eradication of P. aeruginosa planktonic cells (initial concentration 108 cells mL−1) in 24 h compared to a 3‐log reduction caused by UV‐A light alone. In contrast, neither the photocatalytic treatment with TiO2 film nor that with TiO2 nanopowder had any effect on P. aeruginosa biofilms at all the interfaces investigated. Possible explanations for these findings, and for the discrepancy between this work and literature data, are discussed.

Importance of subaerial biofilms and airborne microflora in the deterioration of stonework: a molecular study

The study characterized the sessile microbial communities on mortar and stone in Milan Universitys Richinis Courtyard and investigated the relationship between airborne and surface-associated microbial communities. Active colonization was found in three locations: green and black patinas were present on mortar and black spots on stone. Confocal laser scanning microscopy, scanning electron microscopy and culture-independent molecular methods revealed that the biofilm causing deterioration was dominated by green algae and black fungi. The mortar used for restoration contained acrylic and siloxane resins that could be used by microorganisms as carbon and energy sources thereby causing proliferation of the biofilm. Epifluorescence microscopy and culture-based methods highlighted a variety of airborne microflora. Bacterial and fungal counts were quantitatively similar to those reported in other investigations of urban areas, the exception being fungi during summer (1–2 orders of magnitude higher). For the first time in the cultural heritage field, culture-independent molecular methods were used to resolve the structure of airborne communities near discoloured surfaces, and to investigate the relationship between such communities and surface-associated biofilms.

Evaluation of Zosteric Acid for Mitigating Biofilm Formation of Pseudomonas putida Isolated from a Membrane Bioreactor System

This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (−97%) and thickness (−50%), and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition.

Assessing the microbiological risk to stored sixteenth century parchment manuscripts: a holistic approach based on molecular and environmental studies

The microbial risk for the conservation of seven sixteenth century parchment manuscripts, which showed brown discolouration putatively caused by microorganisms, was evaluated using non-invasive sampling techniques, microscopy, studies of surface-associated and airborne microflora with culture-independent molecular methods, and by measuring repository thermo-hygrometric values. Microscopic observations and ATP assays demonstrated a low level of contamination, indicating that the discolouration was not related to currently active microbial colonisation. Nevertheless, a culture-independent molecular approach was adopted to fully characterise surface-associated communities searching for biodeteriogens that could grow under appropriate thermo-hygrometric conditions. Indeed, potential biodeteriogens and microorganisms that are ecologically related to humans were found, suggesting the need to control the conservation environment and improve handling procedures. Microbial loads of air and thermo-hygrometric measurements showed that the repository was not suitable for preventing the microbial deterioration of parchment. A holistic approach to the assessment of risk of microbial deterioration of documents and heritage preservation is proposed for the first time.

Permeabilization method for in‐situ investigation of fungal conidia on surfaces

Aims:  4′,6‐diamidino‐2‐phenylindole (DAPI) staining and fluorescent in‐situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces.

Culture-Independent Methods to Study Subaerial Biofilm Growing on Biodeteriorated Surfaces of Stone Cultural Heritage and Frescoes

Actinobacteria, cyanobacteria, algae, and fungi form subaerial biofilm (SAB) that can lead to material deterioration on artistic stone and frescoes. In studying SAB on cultural heritage surfaces, a general approach is to combine microscopy observations and molecular analyses. Sampling of biofilm is performed using specific adhesive tape and sampling of SAB and the substrate with sterile scalpels and chisels. Biofilm observations are carried out using optical and scanning electron microscopy. Specific taxa and EPS in biofilm can be readily visualized by fluorochrome staining and subsequent observation using fluorescence or confocal laser scanning microscopy. The observation of cross sections containing both SAB and the substrate shows if biofilm has developed not only on the surface but also underneath. Following nucleic acid extraction, 16S rRNA gene sequencing is used to identify bacterial taxa, while 18S rRNA gene and internal transcribed spacer (ITS) sequence analysis is used to study eukaryotic groups. In this chapter, we illustrate the protocols related to fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), and denaturing gradient gel electrophoresis (DGGE).

A new non-degenerate primer pair for the specific detection of the nitrite reductase gene nrfA in the genus desulfovibrio

Dissimilatory nitrate reduction to ammonia (DNRA) is the process in which nitrate is reduced, via nitrite, to ammonia. Bacteria known to carry out DNRA mainly originate from wastewater treatment plants, where DNRA is a relevant process. The ability to carry out DNRA is phylogenetically widespread, and the gene nrfA, encoding for the key enzyme of the second step of the pathway, could be used as a marker for this process. In this study we developed a new primer pair specific for nrfA in the genus Desulfovibrio. The specificity of the primer pair was tested on DNA from thirteen species of Desulfovibrio and DNA from two wastewater samples. PCR amplifications yielded products of the expected size (850 bp), and sequences obtained from Desulfovibrio strains and environmental sample clone libraries matched the Desulfovibrio nrfA gene. Nevertheless, we found nrfA gene sequences in the environmental samples that are not present in the databases. The new primer set can be used to obtain more sequences of the nrfA gene and improve our knowledge of the DNRA pathway in this genus, e.g. with the aim to improve the wastewater treatment process.