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 3AE5 · 7AE8 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 ÔbiocleaningÕ and ÔbiorestorationÕ 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.

Biofilms constitute the predominant microbial style of life in natural and engineered ecosystems. Facing harsh environmental conditions, microorganisms accumulate reactive oxygen species (ROS), potentially encountering a dangerous condition called oxidative stress. While high levels of oxidative stress are toxic, low levels act as a cue, triggering bacteria to activate effective scavenging mechanisms or to shift metabolic pathways. Although a complex and fragmentary picture results from current knowledge of the pathways activated in response to oxidative stress, three main responses are shown to be central: the existence of common regulators, the production of extracellular polymeric substances, and biofilm heterogeneity. An investigation into the mechanisms activated by biofilms in response to different oxidative stress levels could have important consequences from ecological and economic points of view, and could be exploited to propose alternative strategies to control microbial virulence and deterioration.

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.

The antifoulant, zosteric acid, was synthesized using a non-patented process. Zosteric acid at 500 mg l(-1) caused a reduction of bacterial (Escherichia coli, Bacillus cereus) and fungal (Aspergillus niger, Penicillium citrinum) coverage by 90% and 57%, respectively. Calculated models allowed its antifouling activity to be predicted at different concentrations. Zosteric acid counteracted the effects of some colonization-promoting factors. Bacterial and fungal wettability was not affected, but the agent increased bacterial motility by 40%. A capillary accumulation test showed that zosteric acid did not act as a chemoeffector for E. coli, but stimulated a chemotactic response. Along with enhanced swimming migration of E. coli in the presence of zosteric acid, staining showed an increased production of flagella. Reverse transcriptase-PCR revealed an increased transcriptional level of the fliC gene and isolation and quantification of flagellar proteins demonstrated a higher flagellin amount. Biofilm experiments confirmed that zosteric acid caused a significant decrease in biomass (-92%) and thickness (-54%).

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Recent scientific investigations have shed light on the ecological importance and physiological complexity of subaerial biofilms (SABs) inhabiting lithic surfaces. In the field of sustainable cultural heritage (CH) preservation, mechanistic approaches aimed at investigation of the spatiotemporal patterns of interactions between the biofilm, the stone, and the atmosphere are of outstanding importance. However, these interactions have proven difficult to explore with field experiments due to the inaccessibility of samples, the complexity of the ecosystem under investigation and the temporal resolution of the experiments. To overcome these limitations, we aimed at developing a unifying methodology to reproduce a fast-growing, phototroph-heterotroph mixed species biofilm at the stone/air interface. Our experiments underscore the ability of the dual-species SAB model to capture functional traits characteristic of biofilms inhabiting lithic substrate such as: (i) microcolonies of aggregated bacteria; (ii) network like structure following surface topography; (iii) cooperation between phototrophs and heterotrophs and cross feeding processes; (iv) ability to change the chemical parameters that characterize the microhabitats; (v) survival under desiccation and (vi) biocide tolerance. With its advantages in control, replication, range of different experimental scenarios and matches with the real ecosystem, the developed model system is a powerful tool to advance our mechanistic understanding of the stone-biofilm-atmosphere interplay in different environments.

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.