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Effect of Cleaning on the Distribution of Microorganisms on Rock Surfaces Thomas Warscheid, Karin Petersen & Wolfgang E. Krumbein Geomicrobiology Division, University of Oldenburg, Postbox 2503, 2900 Oldenburg, FRG ABSTRACT Bacteria, fungi, algae and actinomycetes were quantitatively detennined in the uppermost layers of sandstones of several monuments within Germany before and after cleaning the stone surface by using high pressure water blasting. Dilution and inoculation for counts was performed using a spiral plater technique. The results of the study indicate that, depending on the original distribution of microorganisms, the microfiora of sandstones immediately after treatment is not drastically altered, especially with regard to specific groups of microorganisms, which were isolated and counted under oligotrophic conditions. After a short period of time microorganisms again reach their previous number or even exceed it. Cleaning by the conventional methods cannot be regarded as a procedure which reduces microbial colonisation indefinitely. INTRODUCTION In recent years it has been widely demonstrated how microorganisms in addition to physical and chemical influences take part in the weathering of rocks (Webleyet al., 1963), as well as in the destruction of stone in buildings and historical monuments. The pioneering inhabitants of freshly exposed rocks devoid of organic matter are usually photoautotrophic microorganisms accumulating photosynthetically synthesised organic carbon for copiotrophic (in 455 D. R. Houghton et al. (eds.), Biodeterioration 7 © Elsevier Science Publishers Ltd 1988 456 Thomas Warscheid, Karin Petersen, Wolfgang E. Krumbein nature zymogenous) and oligotrophic heterotrophs, which in tum supply sulphur bacteria and nitrifying bacteria with energy sources, such as hydrogen sulphide and ammonia (Krumbein, 1973). However, only in distinct microhabitats are microorganisms able to attack the mineral-surface more vigorously than abiotic aggressive compounds. Beneath the excretion of enzymes, protons, inorganic and organic acids and complex forming agents microorganisms accumulate dissolved cations in their cells leading to a continuously steady state in the weathering process which ends in the destruction ofthe rock-forming mineral (Beveridge, 1984). Nevertheless the mere occurrence of microorganisms on stone surfaces does not automatically imply destructive action. But these rock- populations may be involved in enhancing abiotic processes or lead to the formation of algae and microbial mats, which may in tum build surface crusts, to the aesthetic detriment of historic monuments. In conservation it is common practice to clean stone surfaces by high pressure water blasting in order to remove biogenic layers and crusts and to prepare the stone for further treatment, such as rendering it hydrophobic. This report represents results concerning the effectiveness of this procedure to reduce the numbers of microorganisms attached to the stone surface and subsurface and its influence on the distribution and re- settlement of these microorganisms. MATERIALS AND METHODS Samples Specimens with typical deterioration characteristics of the following monuments were analysed: Marien-Church, Ge1nhausen-Quartzitic Lower Triassic-Sandstone. S I-with algae layer and some crustose lichens on the solid stone surface; S 3-exfoliating stone surface covered with crustose lichens; S 2 and S 4-same as S 1, S 3 reinspected immediately after washing. St. Lorenz-Church, Nuemberg-Quartzitic 'Burg'-Sandstone. S 5-Black crust on solid stone surface; S 7 -thick algae carpet; some crustose lichens on solid stone surface; S 6 and S 8-same as S 5, S 7 reinspected between 1 and 2 weeks after washing. St. Matthias-Church, Trier-Quartzitic Lower Triassic-Sandstone. S 9 with black-shaped algae layer on solid stone surface; S lO-sample site comparable to S 9 about 5 weeks after washing. Distribution of microorganisms on rock surfaces after cleaning 457 'Alte Pinakothek', Munich-carbonaceous Cretaceous-Sandstone (,Gruensandstein,). S II-black crust with underlying crumbly stone surface; S 12-sample site comparable to S 11 about 8 weeks after washing. Sampling Incisions were cut about 2-3 mm deep in an area of3 X 3 cm ofthe stone surface using a drill with a diamond wheel. Samples were removed with a stone chisel. Approximately 109 of material was collected in sterile petri- dishes and analysed one day after collection. Culture media Copiotrophic, chemoorganotrophic bacteria: DEV Gelatine-Agar (Merck, Darmstadt W.G., Nr. 10685). Oligotrophic, chemoorganotrophic bacteria: DEV Gelatine-Agar (same as above, but diluted 1/50) and Bunt & Rovira's medium. Nitrifying bacteria: media submitted by Bock (personal communication). Copiotrophic fungi: Sabauraud 4% Maltose-Agar (Merck, Darmstadt W.G., Nr. 5439). Oligotrophic fungi: Sabauraud 4% Maltose-Agar (as above, but diluted 1/50) and Czapek-Dox-Agar (Merck, Darmstadt W.G., Nr 5460 but diluted 1/50). Algae and cyanobacteria: BG ll-Medium. Actinomycetes: Glycerol-nitrate-casein-agar (but diluted 1/50). Inoculation, incubation and enumeration Sample material was carefully powdered in a hand mortar, suspended in sterile 0·001 % Tween 80-solution (10%; w/v) and shaken continuously for 1 h. Chemoorganotrophic bacteria and fungi as well as actinomycetes were inoculated using the modified spiral plater technique of Lewis et aZ. (1985). Nitrifying bacteria, algae and cyanobacteria were inoculated as prescribed for the MPN method. The inoculated samples were incubated at 28°C in the case of chemoorganotrophic bacteria, fungi, actinomycetes and nitrifying bacteria and at 16°C in light (about 1000 lux) in the case of algae and cyanobacteria. Counting, of the chemoorganotrophic bacteria, fungi and actinomycetes according to the Spiral Plater procedure was carried out 2 weeks after inoculation. Nitrifying bacteria were enumerated 6 weeks, algae and cyanobacteria 8 weeks after inoculation. Counts were computed as CFU/g stone. 458 Thomas Warscheid, Karin Petersen, Wolfgang E. Krumbein RESULTS AND DISCUSSION Counts of isolated microorganisms from the different samples are summarised in Table 1. It can be shown that immediately after the high pressure water blasting treatment on the stone surface (S I-S 4) a short- term impairment of the colonisation of microorganisms is obvious. The species-composition however, remained extensively stable. Oligotrophic bacteria predominate to a greater extent compared with the copiotrophic ones. Fungi, whose differentiation with respect to copiotrophic and oligotrophic characteristics, in this case are less distinct, remain dominant in sample S 1/S 2 and inferior in sample S 3/S 4. Nitrifying bacteria were found preferentially on the sample-site which was highly infected by a chemoorganotrophic micro flora. This site is characterised by microsoil-conditions, arising behind the exfoliating stone surface, including the presence of actinomycetes (Krumbein, 1973; Strzelczyk, 1981). Shortly after the washing procedure (S 5-S 8) the numbers of microorganisms develop differently, depending on their previous colonisation before treatment. The species-composition of organisms remained unchanged to this point, but the diminution of their number on the slightly algae colonised black crust (S 5/S 6) was more lastingly preserved, especially in the case of nitrifying bacteria. Nevertheless the growth of algae is stimulated by the introduced water of the cleaning procedure. In comparison the originally dense algae overgrown sample site S 7 shows a revival of the chemoorganotrophic bacterial flora after washing, which may be due to the fresh and unweathered stone surface, promoting new attachment of microorganisms or the increased algae growth stimulated by humidity. Additionally, the partial conservation ofthe accumulated organic compounds on the stone, supporting microbial growth, by washing them into the interior of the stone has also to be considered regarding the coarse-grained character of the sandstone of this site, compared with the fine-porous character of the sandstone in the samples S l-S 4. Coarse- grained sandstones possess larger pores allowing a high influx and emux of nutrient enriched solutions thereby increasing the growth rate of colonising microorganisms. Some weeks after the washing procedure (S 9/S 10) microorganisms were reactivated even on the fine-grained lower Triassic-Sandstone whereby oligotrophic bacteria again dominate. More drastic was the recurrence of stone surface-infecting micro- organisms on highly calcareous, water absorbing and porous Cretaceous- Sandstone about 8 weeks after the cleaning (S 11/S 12). T A B L E 1 N um be rs o f M ic ro or ga ni sm s on R oc k Su rf ac es (C F U /g s to ne ) o n V ar io us S pe ci m en s T ak en fr om S ev er al M on um en ts w it hi n G er m an y be fo re an d a ft er C le an in g P ro ce du re w it h H ig h P re ss ur e W at er B la st in g S a m p le B ac te ri a F un gi A lg ae A ct in om yc et es C op io tr op hi c O li go tr op hi c N it ri fy in g C op io tr op hi c O li go tr op hi c S 1 90 0 12 00 0 75 00 0 65 50 0 24 00 0 0 8 2 18 0 78 5 40 34 00 0 23 00 0 43 0 0 S 3 31 0 00 0 18 00 0 00 24 00 44 00 34 50 11 0 00 0 43 00 S 4 11 00 00 19 50 00 43 0 0 0 0 0 S 5 11 0 00 10 60 0 15 00 00 51 00 27 00 0 0 0 S 6 45 0 30 00 0 34 00 59 00 75 00 0 S 7 32 00 00 99 00 00 39 0 32 00 39 50 0 55 00 30 00 0 S 8 67 00 00 13 00 00 0 43 0 0 62 50 55 00 00 0 8 9 79 70 0 10 30 00 90 35 00 57 00 12 00 00 0 S lO 12 00 0 22 50 00 0 61 00 27 90 0 0 S I1 12 00 13 00 0 17 00 34 35 20 0 0 S 12 82 00 00 28 50 00 44 00 58 00 66 00 75 00 54 00 t:::l a" ~ ;::: ~. ;:: ~ :i ;::; . ~ c ~ ;:: ~. '" c ;:: ~ '" ;oq- '" ;::: ~ '" ~ .g, ~ ~ ;:: ~. ~ Vl 1.0 460 Thomas Warscheid, Karin Petersen, Wolfgang E. Krumbein All groups of microorganisms analysed reach a large increase in numbers, which may also be due to the freshly exposed stone surface after cleaning as well as to the high porosity of the material offering optimal colonisation conditions for the organisms. Therefore it can be stated that high pressure water blasting only temporarily affects the number of microorganisms on stone surfaces. After a short period, microorganisms recover and either reach their previous number or exceed it drastically, depending on the kind of sandstone in question. After the initial cleaning process, additional conserving treatments, such as rendering the stone hydrophobic, are necessary to enable successful and enduring restoration. Nevertheless it can be seen in recent analyses that even the protective skin of the hydrophobic medium offers new possibilities for the colonisation by different microorganisms. In order to reach a better understanding of the attachment and distribution of microorganisms on stone surfaces further studies will be undertaken. ACKNOWLEDGEMENTS The Federal Minister of Science and Technology has largely contributed to our work through generous grants. D. Janssen has been of great help in our work with technical assistance. REFERENCES Beveridge. T.1. (1984). Proceedings of the 3rd International Symposium on Microbial Ecology, Michigan State University. American Society for Microbia, Washington D.C., pp. 601-7. Krumbein, W.E. (1973). Deutsche Kunst- und Denkmalpflege, 31, 54-71. Krumbein, W.E. & Schonborn-Krumbein, C.E. (1987). Bautenschutz und Bautensanierung, 10, 14-23; 110-17. Lewis, F., May, E. & Bravery, AF. (1985). V'e Congres international sur 1'Alteration et la conservation de la Pie"e, Lausanne. Presses Polytechniques Roman des, Lausanne, pp. 633-42. Strzelczyk, AB. (1981). Stone. Microbial Biodeterioration, ed. AM. Rose. Academic Press, New York, pp. 61-80. Webley, D.M., Henderson, M.E.K. & Taylor, I.F. (l963).Journal of Soil Science, 14 (1), 102-12. An extended bibliography for this paper may be obtained from the senior author.
