## 5 Biocleaning
### Removal of Inorganic Materials--Black Crusts, Nitrate Crusts  
Although some black crusts may be layers of dark pigmented cyanobacteria [3], those most commonly recognized on historic stone buildings are composed of gypsum (calcium sulphate dihydrate) and dark particulates from air pollution [98]. Traditionally, these are removed chemically with an ammonium carbonate-EDTA mixture, which may damage the underlying stone if not carefully applied. In 1992, [PERSON] et al. [99] reported a method for removing these layers from marble using a culture of _Desulfovibrio desulfuricans_, an anaerobic sulfate-reducing species better known for its ability to produce microbially-influenced corrosion of steel structures [100], and references therein). After various efforts to improve the treatment (e.g., [101; 102]), it was shown to be effective in removal of the crusts and preservation of the noble patina beneath them and has now been applied successfully to various monuments (e.g., [103; 104]). Care must be taken, however, with the duration of the treatment. [PERSON] et al. [105] used sulfate-reducing bacteria to remove sulfate crusts and found that prolonged contact between the bacteria and the stone resulted in damaging sulfide precipitation.  
Nitrate crusts are one of the types of stone efflorescences which are often considered to be of particular importance in deterioration of indoor artworks, such as mural paintings. [PERSON] et al. [106] were among the first to suggest the use of nitrate reducing bacteria (a strain of _Pseudomonas stutzeri_ in this case) to remove nitrate salts from stone. There have now been several successful cases of biocleaning of nitrates from stone; the wall paintings in the central vault of Santo Juanes church in Valentia, Spain, and the external wall of Matera cathedral are examples [107]. The latter building was treated with a novel carrier system (Carbogel) containing _Pseudomonas pseudocalalignes_ that was covered with a PET film. Another novel application system was used on the mural paintings in the Santo Juanes church in Valentia [108]; _Pseudomonas stutzeri_ DSMZ 5190 in a Japanese paper and agar layer was heated with infrared to a suitable temperature for optimum bacterial activity. Recently, [PERSON] et al. [109] suggested that extremophilic bacteria might be good candidates for biological removal of efflorescences. Extremophiles live under extreme conditions, very high or very low temperatures, pH values and pressures, or in the presence of normally toxic agents such as irradiation and heavy metals. Often, they belong to the Archaea domain, but they can also be members of the Bacteria or the Eukarya (fungi or algae). The unusual conditions required for their growth means that their application as biocleaning agents should be safe, with no undesirable future effects on the environment or adverse effects on current lifeforms (including humans). This could make them more acceptable for use by conservators and restorers. [PERSON] et al. [109] screened various extremophiles from the culture collection held by CNR, Italy, and selected an aerobic bacterium, _Halomonas campaignis_ 5 AGT, as a potential remediation treatment for nitrate crusts. The bacterium lives at high pH values and is non-pathogenic.  
Mixed salt crusts also occur and these have been removed by combined biological and chemical treatments ([110], and references therein). These crusts may contain not only sulfates and nitrates, but also carbonates, apatite and protein, hence posing a challenge to traditional techniques that could be overcome by these innovative methods.