Biocleaning on Cultural Heritage: new frontiers of microbial biotechnologies.

Over the last two decades, the biotechnologies applied to Cultural heritage (CH) have become a successful novel alternative to the traditional approaches in the CH conservation and preservation. From these new perspectives, microorganisms and their metabolisms can be used for the safeguarding of artworks. Biocleaning is a field with a growing interest, based on eco-friendly processes and safe procedures, where biological reactions occurring in natural habitats are optimized in artificial conditions with the aim of CH conservation. This represents a new tool and opportunity for the development and improvement of the sector, with a great advantage for the CH conservation-restoration, in terms of safety, effectiveness, costs and environmental sustainability. This review focuses on the use of microbes and enzymes involved in biocleaning of CH artworks. The aim is to provide a comprehensive, critical and chronological view of the scientific works published until now where 'virtuous' microorganisms are applied on different CH materials, pointing out strength and drawback of the biocleaning treatments.

Onsite advanced biocleaning system for historical wall paintings using new agar-gauze bacteria gel.

AIMS: This study reports the results of the application of a new agar-gauze biogel system activated with viable bacterial cells to altered wall paintings. METHODS AND RESULTS: Biocleaning using agar biogel and agar-gauze biogel systems was performed onsite by direct application to altered wall painting surfaces (25-1000 cm2 ). The treatments were performed for the restoration of two original Italian sites: (i) at the Vatican Museums, Cristo che salva Pietro dalle acque-La Navicella, a wall painting by Giovanni Lanfranco (1627-1628) and (ii) at Pisa Cathedral Cupola, Incarnato, a wall painting by Orazio Riminaldi (1593-1630) and his brother Girolamo Riminaldi. The novelty of this study is the use of viable Pseudomonas stutzeri A29 cells in an advanced agar-gauze biogel system and the short bio-application contact times of between 3 and 12 h. The historical artworks were altered by lipid and protein residues from past restoration, as confirmed by Py-gas chromatography-mass spectrometry and FT-IR data. The effectiveness of the biological treatment was assessed, and general considerations were discussed. CONCLUSIONS: The short bio-application contact time of advanced agar-gauze gel activated with viable P. stutzeri cells makes this biotechnology promising as an alternative method to the traditional onsite cleaning techniques currently in use for altered historical wall paintings. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we report for the first time the biocleaning of altered materials located in vertical and vaulted areas using agar-gauze biogel with short application times. These findings are of great significance for future restoration activities and are crucial for determining the best preservation strategies in this field.

Hi-tech restoration by two-steps biocleaning process of Triumph of Death fresco at the Camposanto Monumental Cemetery (Pisa, Italy).

AIMS: In this work, the 'hi-tech' complex biocleaning and restoration of the 14th-century fresco Triumph of Death (5·6 × 15·0 m) at the Camposanto Monumental Cemetery (Pisa, Italy) is reported. Since 2000, the restoration based on the biological cleaning of noble medieval frescoes, has been successfully utilized in this site. METHODS AND RESULTS: The novelty of this study is the two-steps biocleaning process using Pseudomonas stutzeri A29 viable cells, previously applied for recovering other valuable frescoes. In this case, after the fresco detachment from the asbestos-cement support (eternity), both the animal glue and the residues of calcium caseinate were biologically removed respectively from the front and from the back of the fresco in 3 h as indicated by GC-MS and PY/GC-MS analyses. The data obtained during the monitoring of the biorestoration process confirmed that the adopted procedure does not leave residual cells on the fresco surfaces as showed by plate count method, ATP determination and also SEM observation. In addition, to avoid the risk of condensation phenomena after the relocation of the restored fresco sections onto the original walls, the use of a new support has been set up together with the design of a control system that allows a continuous monitoring of environmental parameters for prevention and conservation purposes. CONCLUSIONS: This large-scale biorestoration work clearly shows and confirms that this biotechnology is highly efficient, safe, noninvasive, risk-free and very competitive compared to the traditional cleaning methods, offering an unusual 'resurrection' of the degraded artworks also in very complicated and delicate conditions such as the Triumph of Death fresco, defined for its dimension and artistic importance the 'Pisa's Sistina frescoes'. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings can be of significant importance for other future new restoration activities and they are crucial for determining preservation strategies in this field.

Inactivation of wine spoilage yeasts Dekkera bruxellensis using low electric current treatment (LEC).

AIMS: The objective of this study was to investigate the inactivation of a selected yeast Dekkera bruxellensis strain 4481 in red wine by application of low electric current treatment (LEC). METHODS AND RESULTS: LEC (200 mA) was applied for 60 days to a red wine, Montepulciano d'Abruzzo, in an alternative strategy to the SO(2) addition during wine storage. The LEC effect on both cell activity and microflora viability was assessed. LEC decreased significantly the survival viable cells and increased the death rate of D. bruxellensis strain 4481 yeast. A final comparison was made of the main physico-chemical parameters of the wine after the different treatments. The study suggests the importance of an appropriate LEC treatment which limits wine deterioration in terms of off-flavours synthesis. CONCLUSIONS: The results demonstrate that the growth of undesirable Dekkera can be inhibited by low voltage treatment; LEC was shown to be useful to prevent wine spoilage and has the potential of being a concrete alternative method for controlling wine spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Wine spoilage can be avoided by preventing the growth of undesirable Dekkera yeasts, through the effective use of LEC in the winemaking process.

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.

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.

Pile composting of two-phase centrifuged olive husk residues: technical solutions and quality of cured compost.

The present work proposed an economically sustainable solution for composting olive humid husks (OHH) and leaves (OL) at a small/medium sized olive oil mill. We planned and set up a composting plant, the prototype taking the form of a simplified low-cost turning machine, and evaluated the use of an inoculum of one year-old composted humid husks (CHH) and sheep manure (SM) to facilitate the starting phase of the process. Trials were carried out using four piles under different experimental conditions (turnover, static, and type of inoculum). The best results were achieved with turnover and an inoculum that induced fast start-up and a correct evolution of the composting process. The final product was a hygienically clean, cured compost.

Effects of low electric current (LEC) treatment on pure bacterial cultures.

AIMS: This research focused on the effects of low electric current (LEC) on the cell viability and metabolic activity of Escherichia coli and Bacillus cereus. METHODS AND RESULTS: Different LEC intensities at fixed amperage were applied, employing either graphite or copper electrode pairs, and the effects were determined by conventional cultural methods and bioindicators. On E. coli, the LEC with graphite electrodes at 5 and 10 mA led to no significant variation, but at 20 and 40 mA there was increasing inhibition of both the enzymatic activities and growth, and a reduction in ATP content. On B. cereus, similar experiments at the lower amperages did not have any inhibitor effects, however, the 40 mA current stimulated growth, ATP content and some enzymatic activities. The LEC treatment using copper electrodes caused, already at 5 mA, inhibition of bacterial growth and metabolic and enzymatic activities in both E. coli and B. cereus. CONCLUSIONS: On the basis of the obtained results using different amperages and electrodes, we can conclude that E. coli seem to be more sensitive compared with B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on LEC treatment effects on the pure bacterial cultures.

Scaling-up in industrial winemaking using low electric current as an alternative to sulfur dioxide addition.

AIMS: To better understand the outcome of employing low electric current (LEC) technology as a new preservation and alternative in wine technology, and to contribute to its development. It is used in industrial-scale winemaking with commercial yeast (Saccharomyces cerevisiae) during the grape must fermentation. METHODS AND RESULTS: LEC (200 mA, time 16 days) was applied to fresh grape must as an alternative method to the usual sulfur dioxide addition used in the industrial process; two tanks, each 30,000 l, were employed for parallel fermentations. The results show that LEC decreased the survival time and increased the death rate of apiculate yeasts, whereas it did not affect the growth and survival of S. cerevisiae. A comparison was made of the main chemical and sensory parameters of the wines obtained. CONCLUSIONS: The results have demonstrated that the low-voltage treatment had a positive effect on the grape juice fermentation (yeast microflora) during the early stages of winemaking. SIGINIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing, for 'biological wine', new winemaking technologies for an innovative control process of yeast fermentation.

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.

Controlling grape must fermentation in early winemaking phases: the role of electrochemical treatment.

AIMS: To contribute to an understanding of the phenomena related to the effect of low electric current (LEC) in grape must fermentation during laboratory and pilot plant scale winemaking, with selected co-culture yeasts (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermodii strain 465). METHODS AND RESULTS: LEC (10, 30, 50 and 100 mA) was applied to fresh grape must as an alternative method to the usual addition of SO2. Parameters such as polarity, treatment duration (24-96 h) and type of inoculum yeast were varied one at a time. LEC decreased the survival time and increased the death rate of H. guilliermondii strain 465 in co-cultures, whereas it did not affect the growth and survival of S. cerevisiae strain 40. A final comparison was made of the main physico-chemical parameters on wine obtained after the different tests. CONCLUSIONS: The results have demonstrated that the low voltage treatment using a pair of graphite electrodes had a positive effect on grape juice fermentation (yeast microflora) during the early stages of winemaking, even with the potential of being an alternative method to the usual addition of SO2. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing new winemaking technologies for an innovative yeast fermentation control process for 'biological wine'.

Effects of low electric treatment on yeast microflora.

AIMS: To contribute to the understanding of phenomena related to different intensity electric current treatments on the growth and metabolism of selected micro-organisms using laboratory samples of pure and co-cultures (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermondii strain 465). METHODS AND RESULTS: Low electric current (10, 30, 50 and 100 mA) was applied to prepared samples. Parameters, such as polarity, treatment duration (18-48 h) and type of inoculum yeast, were varied one at a time to highlight their cause-effect relationships. The effects on cell activity as well as microflora viability were assessed. Bioindicators capable of describing the phenomena caused by the electric current on the microflora were identified. CONCLUSIONS: Results demonstrated that a low voltage treatment using graphite electrodes had a greater effect on the viable S. cerevisiae strain 404 microflora. There was less bactericidal activity in the S. cerevisiae strain 404 than in the H. guilliermondii strain 465. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be of significant importance in the development of new technological processes in the fields of agriculture and food, particularly new fermenting process controls.

Laboratory-scale trials of electrolytic treatment on industrial wastewaters: microbiological aspects.

Animal, civil and industrial waste matter is a source of potential chemical, microbiological and air pollutants. In populated areas the presence of faecal bacteria and the production of malodorous compounds during waste storage and in the tanks of wastewater treatment plants, can cause concern. The general aim of the work was to study electrolytic waste treatment (recently applied on animal slurry) using low electric current across graphite and copper electrodes, determining its effect on the microflora of sludge, collected from the equalisation basin of an industrial aerobic wastewater treatment plant, and on odour emission abatement. Biochemical and enzymatic indicators like ATP content and a pool of 19 enzymatic activities were tested, comparing them with viable cell counts by traditional microbiological methods, to verify the validity of such indicators in monitoring the electrolytic treatment and to assess their correlation with odour reduction. The preliminary results of our laboratory-scale trials showed that in the presence of inert electrodes, such as graphite, metabolic activity is stimulated, whereas with copper electrodes the ATP content and some enzymatic activities are inhibited quite considerably after only four days, this being accompanied by a marked reduction in odour. Consideration was also given to the total copper released from the electrodes and its recovery using iron electrodes.

Composting of solid and sludge residues from agricultural and food industries. Bioindicators of monitoring and compost maturity.

A study to monitor the composting process, to evaluate the effectiveness of bioindicators for the quality and maturity of cured compost obtained by a mixture of winery residues, sludges from dairies and solid residues from food processing (grape-stalks, grape-dregs, rice husks), was conducted. Composting process lasting five months was monitored by chemico-physical, spectroscopic (FTIR, DTG and DSC), microbiological and enzymatic analyses. Biological activities (ATP, DHA contents and several enzymatic activities), impedance variations (DT) of mixed cultures during growth and potential pathogens (E. coli and Salmonella sp.), were determined. The phytotoxicity tests gave a germination index higher than 90% and no significant genotoxic differences between controls and the compost samples were evidenced. Pathogens were not found on the cured compost that can therefore be satisfactorily used as amendment for agricultural crops. However, no single measurement of a composting process factor, biological, chemical or physical, gave a comprehensive view of the quality of a specific composting. We proposed a tool of bioindicators of potential activity and markers in combination for integrated evaluation of monitoring of composting process and compost quality. The responses of several enzymatic activities were positive and indicative of their favorable use capable to reveal even very small changes within microbial population and activity in test and monitoring of compost programmes.

The influence of hay-packing techniques on the presence of saccharopolyspora rectivirgula.

Environmental factors influencing the growth, distribution and viability of thermophilic actinomycetes, especially Saccharopolyspora rectivirgula as an agent of extrinsic allergic alveolitis in farms workers, were studied. Total microbial count, eumycetes and thermophilic actinomycetes were determined on 96 hay samples, randomly collected, from small prismatic and large cylindrical bales, 30 air samples before and after animal feeding, and various surfaces in two farms located in the province of Reggio Emilia, Italy. The number of thermophilic actinomycetes (potentially responsible for hypersensitivity pneumonitis) was higher in hay samples from large cylindrical bales than in those from small prismatic bales. The structural characteristics of the buildings (barns with stalls, poor ventilation) and the feeding practices (manual handling of hay, constant presence of hay in feedings corridors) contributed to the dispersion of high levels of thermophilic actinomycetes spores (potentially responsible for extrinsic allergic alveolitis). The ventilation system proved to be inadequate in reducing the number of microorganisms present.

[The effects of verapamil on the postischemic changes in the coronary microcirculation: the role of nitric oxide]. / Effetti del verapamil sulle alterazioni postischemiche della microcircolazione coronarica: ruolo dell'ossido nitrico.

BACKGROUND: The aim of this study was to evaluate, in the model of isolated working rat heart, the effects of verapamil on postischemic changes in cardiac mechanical function and microvascular coronary permeability, and the possible role of nitric oxide. METHODS: We used 72 male Wistar rats, weighing 250-300 g, divided into six groups: Group A, hearts perfused with modified Krebs-Henseleit solution (KH); Group B, hearts perfused with KH + verapamil 0.25 microM; Group C, hearts perfused with KH + verapamil 0.5 microM; Group D, hearts perfused with KH + verapamil 1 microM; Group E, hearts perfused with KH + NG-nitro-L-arginine methyl ester (L-NAME) 30 microM; Group F, hearts perfused with KH + verapamil 0.25 microM + L-NAME 30 microM. Hemodynamic parameters, necrosis enzyme release and fluoroscein isothiocyanate-albumin extravasation were evaluated. RESULTS: We observed a clear preservation of cardiac mechanical function and microvascular function in Group B (low dose verapamil) compared to groups A (control), C and D (high dose verapamil); the inhibition of nitric oxide-synthase in the presence of verapamil, obtained in Group F, elicited a loss of myocardial protective effects of verapamil. CONCLUSIONS: Our data suggest that low dose verapamil is effective on postischemic damage reduction and most probably nitric oxide plays a determinant role in this effect.

[Effectiveness of paroxetine in the treatment of refractory vasovagal syncope in young patients]. / Efficacia della paroxetina nel trattamento della sincope vasovagale refrattaria in pazienti giovani.

OBJECTIVES: The aim of the study was to assess whether the well-tolerated serotonin re-uptake inhibitor paroxetine hydrochloride could prevent vasovagal syncope in young patients resistant to or intolerant of previous traditional therapies. BACKGROUND: Serotonergic mechanisms may play a major role in the pathophysiology of neurocardiogenic syncope, and serotonin re-uptake inhibitors have been recently reported to be effective in preventing episodes. METHODS: Forty-one consecutive young patients (13 male and 28 female), aged less than thirty years with recurrent syncope and positive head-up tilt test, and in whom standard therapies with beta-blocking, vagolytic, negative inotropic or mineral corticoid agents were ineffectual, poorly tolerated or contraindicated, randomly received either paroxetine at 20 mg once a day or a placebo. A head-up tilt test was then re-performed after one month of treatment, and the clinical effect was noted over a mean follow-up of 27.1 +/- 6.6 months. RESULTS: The response rates (negative tilt test) after one month of treatment were 57.1 versus 33.3% (p < 0.001) in the paroxetine and placebo groups, respectively. During follow-up, spontaneous syncope was observed in 4 patients (19%) in the paroxetine group and in 12 patients (60%) in the placebo group (p < 0.001). Only one patient (4.8%) asked to be discontinued from the drug for severe recurrent headache. CONCLUSIONS: Paroxetine significantly improved symptoms of young patients with recurrent vasovagal syncope unresponsive to or intolerant of traditional medications and was well tolerated by patients.