Selection of Suitable Microorganisms for the Bioremoval of Graf fi ti

As relatively few studies have addressed the biodeterioration and biocleaning of graffiti, few microorganisms with the potential to degrade graffiti paint have been identified (Table8.2). Therefore, focusing on the selection of suitable microorgan-isms is an important area of study. Candidate microorganmicroorgan-isms can be found in international collections of microorganisms (DSMZ, ATCC, CECT, etc.) or in natural environments.

If researchers wish to use registered microorganisms, these should be selected for their particular characteristics and be purchased and assayed in the laboratory in order to evaluate their capacity to grow and degrade graffiti. Although this is a faster and probably cheaper selection method, some studies have shown that it is more difficult to obtain the most suitable microorganisms. Ranalli et al. (2005) conducted a comparative enzymatic analysis of the bioremoval capacity of a bacterium (Pseu-domonas stutzeri A29) directly isolated from the substance to be removed (aged animal glue from frescoes) and compared with six other commercial strains of the same genera (Pseudomonas) and even species (P. stutzeri). This study revealed that all strains tested were able to synthesize inducible specific enzymes after being grown with the target substance (animal glue). However, the isolated strain (P. stutzeri A29) grew optimally and displayed the highest enzymatic (protease) activity (Ranalli et al.2005). A study of the molecular mechanisms of action in this particular strain revealed a unique enzymatic profile when the bacterium was grown with the glue isolated from the fresco. The enzymes may be a mixture of particular proteases whose combined action enables the complete digestion of the hardened glue present in the frescoes (Antoniolli et al. 2005).

A logical starting point for obtaining microorganisms that can act as biocleaning agents from natural sources is to isolate them from the substance to be removed. This was done, for example, by analysing black crust biofilms, from which different researchers have isolated sulphate reducing bacteria (SRB), hydrocarbon degrading bacteria (HDB) and other microorganisms with hydrolytic activities able to degrade polycyclic aromatic hydrocarbons and fatty acids, among others (Saiz-Jimenez 1993,1997; Fernandes2006; Soffitti et al.2019). In the case of graffiti paint, outdoor

Table8.1Comparisonofgeneralstrategiesforthebioremovalofsalts,organicmatterandgraffitibyusingmicroorganismsfrominorganicsubstrate SuitablemicroorganismsApplicationprotocolsMonitoringReferences Target substance toremoveSelectedstrainsMetabolic actionGrowthDeliverysystemApplication time/T

Microbial, chemical and aesthetical changes Sulphate saltsDesulfovibriovulgarisa subsp.vulgaris Desulfovibriovulgarisa Desulfovibrio desulfuricansa

Sulphate reduction activity

DSMZ63modified medium(An)Immersion Sepiolitea Hydrobiogel.97a Carbogela Mortarandalgi- natebeadsa Arbocela

12–110h/ 17–30CAfter treatmentGaurietal.(1989, 1992);Heselmeyer etal.(1991);Ranalli etal.(1996a,1997, 2000);Cappitellietal. (2005,2006);Polo etal.(2010);Gioventù etal.(2011);Troiano etal.(2014);Alfano etal.(2011) Cellulosimicrobium cellulansaCarbonate solubilizationTSAmedium(Ae)Laponitemicro- packsa24–48h/ 6–37CAfter treatmentMazzonietal.(2014) Nitrate saltsPseudomonas pseudoalcaligenesa Pseudomonas aeruginosaa Pseudomonas desnitrificansa

Denitrification activityNitratebrothmedium (Ae)Sepiolitea Mortarandalgi- natebeadsa Carbogela Hydrobiogel- 97a

24h-1 month/ 18–26C

Aftertreat- mentand 8months and6years

Ranallietal.(1996b) Mayetal.(2008) Alfanoetal.(2011) PseudomonasstutzeriaCottonwoola, agara1.5–3h/ 254CAftertreat- mentand 1month

Bosch-Roigetal. (2010,2013,2019) Organic matterPseudomonasstutzeria Collagenolytic andM9supplementedwith 0.5–1%animalglueCottonwoola , agara ,Laponitea1.5–24h/ 283CAfter treatmentRanallietal.(2005); Antoniolietal.(2005) Lustratoetal.(2012) (continued)

Table8.1(continued) SuitablemicroorganismsApplicationprotocolsMonitoringReferences Target substance toremoveSelectedstrainsMetabolic actionGrowthDeliverysystemApplication time/T

Microbial, chemical and aesthetical changes caseinolytic activityBosch-Roigetal. (2010) Rampazzietal.(2018) Stenotrophomonas maltophiliaa Pseudomonas koreensisa

Proteinolytic activityTSAmedium supplementedwithgel- atine1%(w/v),orR2A mediumsupplemented withgelatine0.4% (w/v)(Ae) Laponitea 24–48h/ 6–37CAfter treatmentMazzonietal.(2014) GraffitiDesulfovibrio desulfuricansNitroesterase activityDSMZ63modified medium(An)Immersion49days/ room temperature

After treatmentGiacomuccietal. (2012) Pantoeasp. Alternariaalternata Arthrobacteroryzae Pseudomonas oryzihabitans Bacillusmegaterium Arthrobacter aurescens Bacillusaquimaris Pseudomonas mendocina Microbacterium

UnknownTrypticsoybroth (TSB), minimalsaltsolution Minimalsaltsolution modifiedwithglucose (10mM)(Ae) Immersion25days/ room temperature After treatmentSanmartínetal.(2016)

oleivorans Gordoniaalkanivorans Pseudomonasstutzeria Aerobacteraerogenesa Comamonassp.a

M9enrichedwithgraf- fitipowder(Ae)Cottonwoola , agara20days/ room temperature

After treatmentSanmartínandBosch- Roig(2019) Aerobacteraerogenes, Bacillussubtilis AmixtureofBacillus sp.,Delftialacustris, Sphingobacterium caeniand Ochrobactrumanthropi Comamonassp. Rubellimicrobium thermophilum, Chelatococcus daeguensis, Escherichiacoli Marinospirillumsp.

UnknownSequentially(4steps): (1)Trypticsoybroth (TSB)during2days (2)1/10strengthtryptic soybroth(TSB)during 5days 3)Completemineral (CM)during10days 4)Completemineral (CM)during10days

Immersion27days/ 30CAftereach stepCattòetal.(2021) Aerobacteraerogenesa Comamonassp.aUnknownCMmediumandM9 enrichedwithgraffiti powdera (Ae)

Agara 14days/ room temperature After treatmentSanmartínandBosch- Roigetal. (unpublishedresults) DSMZ63medium:modifiedbyeliminatinganyironsource;AeAerobicmetabolism,AnAnaerobicmetabolism a Subaerealapplication

Table8.2Comparisonofgeneralstrategiesforthelivemicroorganisms’bioremovalofgraffiti BacterialselectionApplicationmaterials andstrategyAnalysisprotocols References Collection sources/registered microorganisms Naturesources/ isolated microorganisms Glassslide/ immersion strategy

Stone/ subaerial strategyVisual alterationColour variantsSurface changesaChemical modificationsbEnzymatic assays Giacomuccietal. (2012)••••••• Sanmartínetal. (2016)••• SanmartínandBosch- Roig(2019)•••••• Cattòetal.(2021)••••• SanmartínandBosch- Roigetal. (unpublishedresults)

•••••• a Referstomicroscopicobservationanalysis b ReferstoFTIRanalysis

graffiti paintings and the inside of spray paint cans are candidate sources of micro-organisms (Sanmartín et al. 2016). After selection of the natural sources to be analysed, the microbial content can be examined by one of the two different methods: (i) the use of culture-dependent techniques (isolation of cultivable micro-organisms) or (ii) the use of culture-independent techniques (studying both cultiva-ble and non-cultivacultiva-ble microorganisms).

The study of cultivable microorganisms is based on microbiological analysis involving isolation, growth and identification stages. Sampling can be done with sterile swabs moistened with sterile buffer solution or by contact plates with general culture media. Samples should then be cultivated in culture solid plates containing general culture media such as nutrient agar or nutrient broth (Fig.8.2). Morpholog-ically different colonies of microorganisms are then selected and sub-cultured to yield pure colonies. The pure colonies can be identified by classical morphological methods (by microscopic examination and dichotomous identification keys), bio-chemical tests (API tests, for example) and by sequencing methods based on the study of 16S rDNA (for bacteria) and ITS or 18S rDNA (for fungi) gene sequences (DNA extraction and polymerase chain reaction [PCR]) (Polo et al.2010). In the field of graffiti biocleaning, only one molecular biological approach has been used to

Fig. 8.2 (a) Sampling bacteria from new graffiti. (b) Example of plates initially used to screen for colonies found on graffiti and associated environments. (c) Different colonies of microorganisms selected, isolated and sub-cultured to test their potential use as graffiti removal agents. The images are reproduced from Sanmartín et al. (2016)

identify microorganisms from natural sources: nine cultivable bacteria and one fungus were identified as candidates for biocleaning purposes by sequencing of 16S rDNA and fungal ITS regions (Sanmartín et al.2016).

Traditional identification techniques are sometimes tedious and time consuming.

Researchers are currently focusing on new methodological advances such as matrix-assisted laser desorption ionization-time offlight mass spectrometry (MALDI-TOF-MS) to identify cultivable microorganisms. This technique captures the molecules present in the microorganisms by detecting proteins, peptides and lipid ions. It identifies a bacterial spectrum, rather than specific DNA sequences. As the samples do not require pretreatment, the method is therefore more rapid than traditional identification techniques; in addition, no reagents are required, and more than 1000 samples can be analysed per day. For identification, the bacterial spectrum obtained must then be compared with the existing databases. The main limitation of this technique is that few of the databases available include environmental microorgan-isms, and that many of the existing ones are costly to access (Shingal et al.2015).

Massive DNA sequencing represents another important methodological advance in the identification of microorganisms. This approach can be used to study cultiva-ble and non-cultivacultiva-ble microorganisms isolated from natural samples because it enables direct sequencing of native DNA/RNA. This next generation sequencing (NGS) technique can provide a more complete view of the microbial communities associated with an object or substrate (Vilanova and Porcar2020). New generation single molecule nanopore technology sequencing (MinION Oxford Nanopore Tech-nologies) is emerging and can be used for the rapid, simple identification of microorganisms as potential biocleaning agents. The huge amounts of nucleotide sequence data must be analysed and compared with existing databases in order to identify the microorganisms. The advantages of this method are that the analysis of long sequences enables more precise identification, little material is needed and the experimental process is simplified. There is no need to grow the microorganisms or to extract or amplify DNA for sequencing (Feng et al.2015). This method provides more comprehensive information about the microbial populations colonizing a particular substrate (e.g. graffiti) because it produces data on all the microorganisms present in the sample.

A combination of culture-dependent and culture-independent techniques should be used to identify and study microorganisms as potential graffiti biocleaning agents.

After identification, the microorganisms must be grown/cultivated in a particular medium supplied with the target substance as a sole carbon source. The biodegradative capacities should then be analysed, among other factors, in order to finally apply them in practical biocleaning actions (Vilanova and Porcar2020).

3.2 Culture Media and Growth Protocols for the Selected