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.

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.

Single strand conformation polymorphism analysis of PCR-tDNA fingerprinting to address the identification of Bacillus species.

The suitability of tDNA-PCR fingerprinting to identify species of the genus Bacillus was tested on 75 strains. Strains belonging to the same species or the same phylogenetic cluster were correctly grouped. Among B. stearothermophilus strains, different pattern types were found. This could be due to the unclear taxonomic situation of these strains, rather than to a failure of the tDNA-PCR. Single strand conformation polymorphism (SSCP) of the PCR products allowed species discrimination within the 'B. subtilis group', but not within the 'B. cereus group'. The tDNA-PCR, alone or coupled with SSCP analysis, is useful to address Bacillus species identification, particularly for those species which are not phylogenetically tightly clustered.

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains.

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains.

Interspecific, intraspecific and interoperonic variability in the 16S rRNA gene of methanogens revealed by length and single-strand conformation polymorphism analysis.

Thirty-seven strains of mesophilic and thermophilic methanogenic Archaea, belonging to 30 species, were analyzed by length polymorphism (LP) and single-strand conformation polymorphism (SSCP) of an amplified 300-bp fragment of the 16S rRNA gene (Escherichia coli positions 9-331) including the variable regions V1 and V2, LPs and SSCPs were detected between species and between strains of the same species (Methanobacterium formicicum). LPs were found in Mb. formicicum DSMZ 3637, Mb. ivanovii DSMZ 2611, Mb. wolfei DSMZ 2970, Methanosarcina barkeri DSMZ 800, and Methanosaeta concilii DSMZ 3671, suggesting the presence of polymorphic 16S rRNA genes in the genome. We propose that LP and SSCP analysis of the 16S rRNA gene could be of practical help for strain identification.

16S-23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus.

The internal transcribed spacers between the 16S and the 23S ribosomal RNA genes were used to discriminate species of the 16S rRNA group I of the genus Bacillus by PCR. The spacer-PCR fingerprints clearly discriminated the different species, except those closely related like the members of the 'B. cereus group' (B. cereus, B. thuringiensis and B. mycoides) and the species of the 'B. subtilis group' (B. amyloliquefaciens and B. licheniformis). Examining in more detail the shortest internal transcribed spacers, B. subtilis group species were distinguished by single-strand conformation polymorphism analysis, whereas B. mycoides was differentiated from B. cereus/B. thuringiensis by restriction analysis.

Aromatic hydrocarbon degradation patterns and catechol 2,3-dioxygenase genes in microbial cultures from deep anoxic hypersaline lakes in the eastern Mediterranean sea.

Several mixed cultures able to grow on different aromatic hydrocarbons were obtained from different depths (between 3500 and 3660 m under the sea surface) of water/brine interfaces (1 to 5 m over the estimated brine surface) of three deep hypersaline anoxic basins (Urania, Discovery and Atalante) in the eastern Mediterranean sea. Eight strains which completely removed toluene from the medium in six to 10 days were isolated from one of the mixed cultures obtained from the Urania basin. The strains grew on toluene and yeast extract in the presence of NaCl concentrations of up to 50 and 100 g l(-1), respectively, indicating that they are halotolerant rather than halophilic. Even though DNA fingerprinting methods showed that the strains were strictly related, two groups could be found on the basis of the plasmid profile. Metabolic profiling and partial sequencing (350 bp) of the 16S rDNA showed that the strains were related to Pseudomonas mendocina. A 320 bp fragment of the catechol 2,3-dioxygenase gene from all the strains was aimplified by PCR. The sequence of the fragment showed 100% identity with xylE from pWW53 of Pseudomonas putida MT53 isolated from soil. Southern hybridisation experiments showed that catechol 2,3-dioxygenase is plasmid encoded.

A comparison among different methods for evaluating the biomass activity in activated sludge systems: preliminary results.

In order to improve activated sludge plant operation (achieving higher efficiency and cost savings) beside influent and effluent characteristics and working parameters (e.g. dissolved oxygen, total and volatile suspended solids, pH, recirculation flow rate, etc.), the biomass activity should be monitored, the bacteria being responsible for the pollutant degradation. Since conventional cultivation based methods are inadequate to quantify environmental microorganisms (due to scarce number of cultivable microorganisms and time-consuming procedures) several "non-conventional" techniques were applied in this study, in order to compare the obtainable information and their routine feasibility. Different measurements (VSS concentration, Oxygen Uptake Rate, microbial counts by cultural and biomolecular methods--MPN-PCR, ATP content, dehydrogenase activity, microbial cell viability and enzymatic activity) were carried out on mixed liquor samples, taken from a municipal activated sludge plant (440,000 p. e.). The preliminary results of the research are presented in this paper.

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.

The microbial degradation of azimsulfuron and its effect on the soil bacterial community.

AIMS: Azimsulfuron is a recently introduced sulfonylurea herbicide useful in controlling weeds in paddy fields. To date very little information is available on the biodegradation of this pesticide and on its effect on the soil microbial community. The aim of this work was to study its biodegradation both in slurry soil microcosms and in batch tests with mixed and pure cultures. METHODS AND RESULTS: Azimsulfuron was applied to forest bulk soil in order to study its effect on the structure of the bacterial soil community, as detectable by denaturant gradient gel electrophoresis (DGGE) analyses. Biodegradation and abiotic processes were investigated by HPLC analyses. In addition, a microbial consortium was selected, that was able to use azimsulfuron as the sole energy and carbon source. One of the metabolites produced by the consortium was isolated and identified through LC-MS analyses. Cultivable bacteria of the consortium were isolated and identified by 16S rDNA sequencing (1400 bp). CONCLUSIONS: Azimsulfuron treatment seems to have the ability to cause changes in the bacterial community structure that are detectable by DGGE analyses. It is easily biodegraded both in microcosms and in batch tests, with the formation of an intermediate that was identified as 2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide. SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on the biodegradation of azimsulfuron and its effects on the soil microbiota.

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.

Metabolism of biphenyl. 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate: the meta-cleavage product from 2,3-dihydroxybiphenyl by Pseudomonas putida.

1. 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid was isolated and identified from washed suspensions of Pseudomonas putida incubated in the presence of 2,3-dihydroxybiphenyl. 2. Benzoic acid was isolated from reaction mixtures of crude cell-free extracts incubated with 2,3-dihydroxybiphenyl. 3. The presence in the same reaction mixtures of either 4-hydroxy-2-oxovalerate or 2-hydroxypenta-2,4-dienoate was suggested by mass spectrometry. 4. The degradative pathway of biphenyl is discussed.

Metabolism of quaternary carbon compounds: 2,2-dimethylheptane and tertbutylbenzene.

Two Achromobacter strains capable of utilizing 2,2-dimethylheptane or tertbutylbenzene as the sole carbon and energy source were isolated from waste-water. Pivalic acid was found in the cultures of Achromobacter A1 containing 2,2-dimethylheptane. From cultures of Achromobacter A2 in the presence of tertbutylbenzene, a diol was isolated and identified as 2,3-dihydro-2,3-dihydroxytertbutylbenzene. Evidence for meta cleavage of the aromatic ring and for accumulation of pivalic acid in the cultures was also obtained. A metabolic pathway for tertbutylbenzene is suggested.

A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis.

Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.

PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis [corrected].

Genomic diversity in 21 strains of Bacillus cereus and 10 strains of Bacillus licheniformis was investigated by random amplified polymorphic DNA (RAPD) analysis, which samples the whole genome, and by two PCR fingerprinting techniques sampling the hypervariable spacers between the conserved 16S and 23S rRNA genes of the rRNA gene operon (ITS-PCR) and regions between tRNA genes (tDNA-PCR). RAPD analysis showed a remarkable diversity among strains of B. cereus that was not observed with the rRNA and tRNA intergenic-spacer-targeted PCR, where all the strains showed practically identical fingerprints. A wide variability among the B. cereus strains was also observed in the plasmid profiles, suggesting that the genetic diversity within B. cereus species can arise from plasmid transfer. One contribution to the diversity detected by RAPD analysis was determined by the presence of large extrachromosomal elements that were amplified during RAPD analysis as shown by Southern hybridization experiments. In contrast to the strains of B. cereus, the 10 strains of B. licheniformis were grouped into two clusters which were the same with all the methods employed. The 16S rRNA genes were identical in all 10 strains when examined using single strand conformation polymorphism analysis after digestion with Alul and Rsal. From these data we hypothesize two different evolutionary schemes for the two species.

Biodiversity of Geodermatophilaceae isolated from altered stones and monuments in the Mediterranean basin.

An investigation was made into the occurrence and biodiversity of Geodermatophilaceae on 78 samples of altered stone surfaces from 24 monuments and natural stones in the Mediterranean basin; it was found that the total microbial counts ranged between 0 and 10(7) cfu g(-1) dry weight. Members of the Geodermatophilaceae family were isolated from 22 of the 78 samples examined, with the incidence of Geodermatophilaceae colonies in the cultivable population ranging from 1% to 100%. The highest percentage was found in six samples of markedly deteriorated stone. Sixty-five strains randomly isolated from the plates were clustered in six different groups by amplified 16S rDNA restriction analysis (ARDRA) using five different restriction enzymes. Twenty-five strains, representing all the ARDRA haplotypes, were characterized further by partial sequencing (350-550 bp) of the 16S rDNA and by analysing 76 morphological, metabolic and physiological properties. The strains were associated with three well-separated clusters of the genera Geodermatophilus, Blastococcus and Modestobacter. On the basis of 16S rDNA sequence and ARDRA analysis, only two strains were found to be related to the two reference strains of Geodermatophilus. All the others could be grouped with Blastococcus aggregatus (19 strains) or the Antarctic species Modestobacter multiseptatus (44 strains), suggesting that it is these two groups, rather than Geodermatophilus, that tend to colonize the stone surfaces, and that Modestobacter-like strains are also found in temperate/Mediterranean climates. From the BOX-polymerase chain reaction (PCR) data, it can be seen that the Modestobacter-like strains, belonging to the most represented ARDRA haplotype (haplotype B, 34 strains), are very polymorphic and that, over a stone surface, there is a wide genetic diversity at the microsite level.

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.