A desiccated dual-species subaerial biofilm reprograms its metabolism and affects water dynamics in limestone.

Understanding the impact of sessile communities on underlying materials is of paramount importance in stone conservation. Up until now, the critical role of subaerial biofilms (SABs) whether they are protective or deteriorative remains unclear, especially under desiccation. The interest in desiccated SABs is raised by the prediction of an increase in drought events in the next decades that will affect the Mediterranean regions' rich stone heritage as never before. Thus, the main goal of this research is to study the effects of desiccation on both the biofilms' eco-physiology and its impacts on the lithic substrate. To this end, we used a dual-species model system composed of a phototroph and a chemotroph to simulate biofilm behavior on stone heritage. We found that drought altered the phototroph-chemotroph balance and enriched the biofilm matrix with proteins and DNA. Desiccated SABs underwent a shift in metabolism to fermentation and a decrease in oxidative stress. Additionally, desiccated SABs changed the water-related dynamics (adsorption, evaporation, and wetting properties) in limestone. Water absorption experiments showed that desiccated SABs protected the stone from rapid water uptake, while a thermographic survey indicated a delay in water evaporation. Spilling-drop tests revealed a change in the wettability of the stone-SAB interface, which affected the water transport properties of the stone. Finally, desiccated SABs reduced stone swelling in the presence of water vapor. The biodeteriorative and bioprotective implications of desiccated SABs on the stone were ultimately assessed.

Zosteric acid and salicylic acid bound to a low density polyethylene surface successfully control bacterial biofilm formation.

The active moieties of the anti-biofilm natural compounds zosteric (ZA) and salicylic (SA) acids have been covalently immobilized on a low density polyethylene (LDPE) surface. The grafting procedure provided new non-toxic eco-friendly materials (LDPE-CA and LDPE-SA) with anti-biofilm properties superior to the conventional biocide-based approaches and with features suitable for applications in challenging fields where the use of antimicrobial agents is limited. Microbiological investigation proved that LDPE-CA and LDPE-SA: (1) reduced Escherichia coli biofilm biomass by up to 61% with a mechanism that did not affect bacterial viability; (2) significantly affected biofilm morphology, decreasing biofilm thickness, roughness, substratum coverage, cell and matrix polysaccharide bio-volumes by >80% and increasing the surface to bio-volume ratio; (3) made the biofilm more susceptible to ampicillin and ethanol. Since no molecules were leached from the surface, they remained constantly effective and below the lethal level; therefore, the risk of inducing resistance was minimized.

Effects of sublethal doses of silver nanoparticles on Bacillus subtilis planktonic and sessile cells.

AIMS: Due to their antimicrobial activity, silver nanoparticles (Ag-NPs) are being increasingly used in a number of industrial products. The accumulation of Ag-NPs in the soil might affect plant growth-promoting rhizobacteria and, in turn, the plants. We describe the effects of Ag-NPs on the soil bacteria Azotobacter vinelandii and Bacillus subtilis. METHODS AND RESULTS: In growth-inhibition studies, A. vinelandii showed extreme sensitivity to Ag-NPs, compared to B. subtilis. We investigated the effects of Ag-NPs at subinhibitory concentrations, both on planktonic and sessile B. subtilis cells. As determined by 2,7-dichlorofluorescein-diacetate assays, Ag-NPs increase the formation of reactive oxygen species in planktonic cells, but not in sessile cells, suggesting the activation of scavenging systems in biofilms. Consistently, proteomic analysis in B. subtilis Ag-NPs-treated biofilms showed increased production of proteins related to quorum sensing and involved in stress responses and redox sensing. Extracellular polysaccharides production and inorganic phosphate solubilization were also increased, possibly as part of a coordinated response to stress. CONCLUSIONS: At low concentrations, Ag-NPs killed A. vinelandii and affected cellular processes in planktonic and sessile B. subtilis cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Re-direction of gene expression, linked to selective toxicity, suggests a strong impact of Ag-NPs on soil bacterial communities.

RNA-based molecular survey of biodiversity of limestone tombstone microbiota in response to atmospheric sulphur pollution.

UNLABELLED: Outdoor stoneworks sustain biofilm formation and are constantly at risk of deterioration by micro-organisms. In this study, the biofilm microflora of historic limestone tombstones located in a highly polluted urban environment (Cambridge, MA) and in a less polluted location (Lexington, MA) were compared using comprehensive RNA-based molecular analyses of 16S rRNA gene sequences as well as sequences of genes for different pathways of sulphur metabolism (soxB, apsA, dsrA). The metabolically active micro-organisms detected by denaturing gradient gel electrophoresis analysis of 16S rRNA fragments were predominantly represented by cyanobacteria (belonging to the family Nostocaceae and to the genus Chroococcidiopsis) in both polluted and unpolluted environments. The investigation of soxB, apsA, dsrA transcripts reflected the abundance and the diversity of sulphur-oxidizing and sulphate-reducing bacteria in the Cambridge samples in comparison with the Lexington samples. The investigation revealed that in addition to phototrophic sulphur bacteria belonging to the genera Thiocapsa, Halochromatium, Allochromatium, Thiococcus and Thermochromatium, other sulphate-oxidizing prokaryotes (e.g. the genus Thiobacillus) as well as sequences of Deltaproteobacteria from the genus Desulfovibrio occurred at the polluted urban site. The interactions between the main functional groups retrieved from the limestone tombstones were discussed. SIGNIFICANCE AND IMPACT OF THE STUDY: The biofilm microflora inhabiting historic limestones are a multi-component open ecosystem sensitively reacting to all environmental factors including air pollutants. Little is known about specific target groups that are active in the biofilm and their physiological functions. For the first time, transcripts involved in important energy-yielding processes were investigated to reveal the metabolic capabilities of the microflora in response to atmospheric sulphur pollution. This work provides novel and important information about the ecology of limestone tombstone microbiota and its complex interaction with the external environment.

Degradation of nitrocellulose-based paint by Desulfovibrio desulfuricans ATCC 13541.

Nitrocellulose is one of the most commonly used compounds in ammunition and paint industries and its recalcitrance to degradation has a negative impact on human health and the environment. In this study the capability of Desulfovibrio desulfuricans ATCC 13541 to degrade nitrocellulose as binder in paint was assayed for the first time. Nitrocellulose-based paint degradation was followed by monitoring the variation in nitrate, nitrite and ammonium content in the culture medium using Ultraviolet-Visible spectroscopy. At the same time cell counts and ATP assay were performed to estimate bacterial density and activity in all samples. Infrared spectroscopy and colorimetric measurements of paint samples were performed to assess chemical and colour changes due to the microbial action. Microscope observations of nitrocellulose-based paint samples demonstrated the capability of the bacterium to adhere to the paint surface and change the paint adhesive characteristics. Finally, preliminary studies of nitrocellulose degradation pathway were conducted by assaying nitrate- and nitrite reductases activity in D. desulfuricans grown in presence or in absence of paint. We found that D. desulfuricans ATCC 13541 is able to transform nitrocellulose as paint binder and we hypothesised ammonification as degradation pathway. The results suggest that D. desulfuricans ATCC 13541 is a good candidate as a nitrocellulose-degrading bacterium.

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.

Color measurements as a reliable method for estimating chlorophyll degradation to phaeopigments.

The application of biocides is a traditional method of controlling biodecay of outdoor cultural heritage. Chlorophyll degradation to phaeopigments is used to test the biocidal efficacy of the antimicrobial agents. In the present study, the usefulness of color measurements in estimating chlorophyll degradation was investigated. An aeroterrestrial stone biofilm-forming cyanobacterium of the genus Nostoc was chosen as test organism, comparing its different behaviour in both planktonic and biofilm mode of growth against the isothiazoline biocide Biotin T®. Changes in A(435 nm)/A(415 nm) and A(665 nm)/A(665a nm) and in the chlorophyll a and adenosine triphosphate (ATP) cell content were compared with the variations in the CIELAB color parameters (L*, a*, b*, C*(ab) and h(ab)). Our findings showed that both the phaeophytination indexes are useful in describing degradation of chlorophyl a to phaeopigments. Moreover, the CIELAB color parameters represented an effective tool in describing chlorophyll degradation. L* CIELAB parameter appeared to be the most informative parameter in describing the biocidal activity of Biotin T® against Nostoc sp. in both planktonic and biofilm mode of growth.

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.

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. METHODS AND RESULTS: We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and beta-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples. CONCLUSIONS: The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.

Detection and elimination of cyanobacteria from frescoes: the case of the St. Brizio Chapel (Orvieto Cathedral, Italy).

A rosy discoloration partly masking the Luca Signorelli frescoes in St. Brizio Chapel (Orvieto Cathedral, Italy) for many years proved to be a biological alteration, so the present research focused on investigating biodeteriogens and selecting an appropriate biocide to treat them. Optical epifluorescence and electronic microscopic observations of the rosy powder revealed a prevalent autofluorescent coccoid form with a diameter bigger than 5 microm. Chlorophylls a and b were extracted, suggesting the presence of cyanobacteria, a thesis subsequently confirmed by flow cytometry. Cultural media were inoculated with the rosy powder, and microorganisms grew as a green patina in phototrophic conditions and as a rosy patina when organic compounds were added to the mineral medium. The rosy discoloration was most likely caused by the presence of phycoerythrin. The sequencing of the cyanobacteria-specific polymerase chain reaction (PCR)-DGGE bands matched, with a similarity percentage >94, uncultured cyanobacteria, and the sequences were deposited in the GenBank under EU874241, EU874242, EU874243, EU874244, EU874245, EU874246, and EU874247. Finally, the efficiency of the two biocides Neo Desogen and Metatin 5810-101, both based on benzalkonium chloride, was evaluated using adenosine triphosphate measurements and PCR-based detection of cyanobacteria. Metatin, used in situ at 2% of the trade product, proved to be the better biocide, no cyanobacteria being detected after the Metatin treatment.

From papyrus to compact disc: the microbial deterioration of documentary heritage.

Highly significant evidence of the intellectual and cultural efforts of the human race is contained in documents. They take many forms, from papyri through paper to modern magnetic media and optical records. These items are mainly made of organic materials many of which contain polymers, which span from cellulose and its derivatives to synthetic resins. As with other manmade objects, however, documentary heritage is susceptible to chemical, physical, and biological damage. For the colonization and establishment of any biological community, the composition of materials used, their status of conservation, and environmental and climatic factors, such as temperature and humidity, are important elements to take into account. This article covers the scientific investigation of microbial degradation of documents, which is one of the most serious and underappreciated sources of damage to library and archival materials. In particular, although less known, modern records, including compact discs, are also subjected to biodeterioration. Archival and library material preservation broadly encompasses those activities and functions designed to produce a suitable and safe environment that extends the life of collections in useable condition for as long as is feasible. In the literature quoted, key information is also provided to avoid or limit microbial growth and some conservation treatments are also reported.

Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes.

AIMS: To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes. METHODS AND RESULTS: The bioremediation intervention consisted of the direct application onto an artwork surface of whole bacterial cells of the Pseudomonas stutzeri A29 strain (bioaugmentation), followed by, in a final step, a purified Protease enzyme. The bioremediation was performed on a Spinello Aretino fresco that had become altered by the animal glue residues of past restoration. For the reader's interest the fresco is the 14th century Conversione di S. Efisio e battaglia (Conversion of S. Efisio and battle), size 3.5 x 7.8 m at the Pisa Camposanto Monumentale, Italy. An assessment was made of the final costs of the biological tests (whole bacterial cells, enzymes) so as to compare them with other intervention techniques. CONCLUSIONS: A successful innovative biological approach to recover valuable frescoes was set up, and the best conditions for treatment efficiency were identified. Furthermore the cost of the biological cleaning using viable bacterial cells and enzymes (P. stutzeri, Protease, Collagenase, 1 : 3 : 10, ratio respectively) was much lower than that of other conventional methods, making this biotechnology not only very interesting but also very competitive. SIGNIFICANCE AND IMPACT OF THE STUDY: New biotechnologies with an innovative, soft approach to the 'biocleaning' and 'biorestoration' of cultural heritage are in constant demand, and our results are clear evidence that such an approach has been achieved; the technique could be of significant importance towards developing other goals.