Deliverables Stone sample information sheets STONE INFORMATION STUDY SHEETS create an extensive database of information about stones showing crust and salt accumulation in buildings of cultural heritage in different climatic zones. INFORMATION DATABASE OF STONES SUITABLE FOR BIOREMEDIATION COVERS: Stones from 13 monuments, mainly in Latvia, Greece and Italy; 22 natural stones; Site accessibility and availability; Mineralogical profiles of crusted stone; Analysis of physical properties beneath crusts; Chemical composition of crusts; Microbiological analysis; Climate and pollution data. CRUST ANALYSIS For physical properties: remarkable shifts towards greater porosity within the weathered uppermost layers of the rocks and plaster, which are ideal for bioremedial techniques. For chemical composition: Sulphates were often present in a narrow 40 mm surface zone of stone blocks. Considerable amounts of sulphate in stone from Matera Cathedral, Riga Brethrens cemetery, Lübeck townhall and Melos. Concentrations at Matera are much more variable than nitrate. Nitrates formed a much more limited gradient on the surface zones. Could only be detected at Matera Cathedral and Riga Brethrens cemetery. For salt concentrations: great variability, can cause major difficulties in bioremediation treatments. For organic pollutants: very low levels, not suited to bioremediation by use of organic degraders. New mortar system for application of nitrate-reducing bacteria to stone A new BioBRUSH mortar was developed as a delivery system for bioremediation work with denitrifying bacteria. It needed a shorter time period for carbonation due a lower content of Portland cement clinker and the addition of pumice and air entraining agent. Alginate was used to immobilise denitrifying bacteria in the mortar and act as a protective cover against alkaline pH, dryness and mechanical stress and ensured homogenous distribution of bacteria in the mortar. Long term frozen storage of previously produced bioactive alginate beads was possible. The optimized mortar delivery system was developed to improve conditions essential for the activity of denitrifying bacteria. Risk evaluation for bioremediation of stone UNDERSTANDING OF RISKS FOR BIOREMEDIATION: Mineralogical change: some superficial anomalies due to deposition of amorphous material may occur but no serious destruction, except with very aged marble which shows some destruction of crystal structures and less cohesive properties. Preliminary tests before, and/or desalination tests after, bioremediation, may be necessary. Visual and aesthetic change: for lighter stones, it was difficult to observe any surface change by biocalcifying bacteria. Darker stones may have lightening of the surface but surface texture was not affected. Some surfaces appeared darker after treatment but the change was not significant. High colour variation for mortar-treated areas with some increased whiteness. Bacteria in delivery system appear to have little effect on appearance, but excessively high densities of biocalcifying bacteria result in visible colour change on stone and produce a thick cracked layer. No significant colour change and no evidence of microorganisms over the long-term. Physical properties: mortar and sepiolite may increase water uptake and porosity of limestone; Carbogel may increase water uptake and porosity or have no effect; sepiolite tends to increase water uptake and porosity; carriers do not seem to affect Gotland Sandstone. Biocalcifying bacteria decrease water absorption in Portland limestone by 1% and decrease open porosity by about 5%. Highly porous stones may not be suitable for bioremediation since salts diffuse back rapidly. Microbial counts: actually higher in untreated areas than treated areas. No long-term consequences leading to growth of microorganisms on stone due to residual medium. Microorganism Data Sheets BIOBRUSH MICROORGANISM DATA SHEETS FOR BIOREMEDIATION AGENTS recorded: Source; Biochemical and cultural characteristics; Biorenediation activities; Identification by molecular sequencing; Level of health risk. Bibliographic searches were done on the three different physiological bacterial groups and this was presented in separate volumes covering: Sulphate-reducing bacteria (SRB); Nitrate-reducing bacteria (NRB); Biocalcifying bacteria. Biocalcifying bacteria were acquired from culture collections and 70 cultures were isolated from an original source in a stream in Somerset, UK. These were screened for their ability to deposit calcite in solid and liquid media. Sulphate removal efficiency: Different 15 selected SRB strains were tested. Nitrate removal efficiency: Nitrate-reducing bacteria were isolated from limnic, marine and various soil environments in order to obtain salt-tolerant microbial strains. Testing 12 out of 36 pure cultures from different environmental sources were found to be highly active. PAH degradation: Brevundimonas diminuta was identified but the organic matter in stone was mostly too low to require biological treatment and studies were discontinued. Methodology for application of biocalcifying bacteria to stone Organism: Pseudomonas putida; Delivery: Apply bacteria direct to stone by brushing (Carbogel prepared with B4-AW medium using Tris-HCL buffer); Cell count: 100 million cells/ml; Medium: B4-AW medium; Special preparations: Apply moistened Japanese paper over bacteria; Application procedure: Apply 1-1.5 cm thickness Carbogel with medium (cover with polyethylene); Application time: 2 weeks. Methodology for application of nitrate- reducing bacteria to stone METHODOLOGY FOR NITRATE REMOVAL USING BIOREMEDIATION Organism: Pseudomonas pseudoalcaligenes; Delivery: CEM mortar, with bacteria immobilised in alginate beads; Cell count: 100 million cells/ml; Medium: DNT medium; Special preparations: Delivery system onto Japanese paper; Application procedure: Apply 1-1.5 cm thickness delivery system (cover with polyethylene); Application time: 1 month. Culture collection for bioremediation THE BIOBRUSH CULTURE COLLECTION was assembled from different sources supported by information about efficacy and safe use in bioremediation. Screening covered biocalcification, hydrocarbon breakdown, denitrification and sulphate reduction. Cultures were also isolated from new sources. The information has been condensed into a database resource as Microorganism Data Sheets. Studies were done on sulphate-reducing bacteria (SRB), nitrate-reducing bacteria (NRB) and biocalcifying bacteria and Microorganism Data Sheets recorded cultural characteristics, activities, identification by molecular sequencing and level of health risk. Screening for calcite production yielded F2 for field trials, identified as Pseudomonas putida, showing low risk to humans, sensitivity to most antibiotics and precipitating calcite at almost all temperatures tested. Brevundimonas diminuta was identified as a potential organic degrader but the organic compounds in stone was mostly too low to require biological treatment so studies were discontinued. For sulphate removal, Desulfovibrio vulgaris subsp. vulgaris was chosen for field trials because of activity under non-strictly anaerobic conditions and ease of detection. For nitrate removal, two NRB environmental isolates, identified as Pseudomonas pseudoalcaligenes and Pseudomonas pertucinogena were chosen, active up to pH 10, at temperatures 10 - 15°C and having no pathogenic properties. Methodology for application of sulphate-reducing bacteria to stone METHODOLOGY FOR SULPHATE REMOVAL USING BIOREMEDIATION Organism: Desulfovibrio vulgaris subsp. vulgaris; Delivery: Carbogel, suspended in SRB medium under anaerobic conditions (bacteria added as washed suspension); Cell count:100 million cells/ml; Medium: DSMZ 63 medium, modified without sulphate or resazurin; Special preparations: Prepare medium without iron and filter to avoid blackening; Application procedure: Apply moistened Japanese paper and then 1-1.5 cm thickness delivery system (cover with polyethylene and exclude as much air as possible); Application time: Maximum 15 hours each application (Treat at least 3 times over 48 hours with Carbogel + SRB end when visually satisfactory). 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