Monument Future

Conclusions

This study compares two different heat scenarios which are both necessary to investigate fire damages on sandstone objects or monuments.

The realistic fire scenario with the exposure of architectural sandstone elements to a burning wood crib within a fire container for a short time results in damages comparable to those observed on monuments which suffered from fire attack. The temperatures measured on stone surfaces and within the inner core of the objects indicate high gradients, resulting in material tension and subsequent cracking.

In contrast, smaller specimens of the same sandstone materials reveal no cracking even at higher temperatures when gradually heated in a laboratory oven. However, such tests and the respective test specimens which will be further investigated, may also be useful for enlightening the change of petrographic and material properties during heating.

Acknowledgements

The authors thank the Free State of Saxony (Sächsische Aufbaubank – Förderbank – SAB) for funding the work within the project WI631.

References

Chakrabarti B., Yates T., Lewry A. 1996. Effect of fire damage on natural stonework in buildings. Construction and Building Materials 10(7):539–544.

Ehling A., Köhler W. 2000. Fire damaged natural building stones. Proc. 6th Int. Congr. on Applied Mineralogy ICAM 2:975–978. Göttingen.

Gómez-Heras M., Álvarez de Buergo M., Fort R., Hajpál M., Török Á., Varas M. J. 2006. Evolution of porosity in Hungarian building stones after simulated burning. In: Fort R., Álvarez de Buergo M., Gómez-Heras M., Vazquez-Calvo C. (eds) Heritage, Weathering and Conservation. Taylor & Francis, London, 513–519.

Gómez-Heras M., McCabe S., Smith B. J., Fort R. 2009. Impacts of Fire on Stone-Built Heritage. Journal of Architectural Conservation 15(2):47–58.

Grunert S. 2007. Der Elbsandstein: Vorkommen, Verwendung, Eigenschaften. Geologica Saxonica – Journal of Central European Geology 52/53:3– 22.

Grunert S., Szilaghy J. 2010. Petrophysikalische Eigenschaften einer Auswahl von Baugesteinen aus Deutschland und ihr Bezug zur Petrographie dieser Gesteine. Geologica Saxonica – Journal of Central European Geology 56(1):39–82.

Hager I. 2014. Sandstone colour change due to the high temperature exposure. Advanced Materials Research 875-877:411–415.

Hajpál M., Török Á. 2004. Mineralogical and colour changes of quartz sandstones by heat. Environmental Geology 46:311–322.

Koser E., Althaus E. 1999. Brandschäden an Bauwerken aus Naturstein – Hohenrechberg und andere Objekte im Laborexperiment. Internationale Tagung des SFB 315 Heft 16/1999.

Lintao Y., Marshall A. M., Wanatowski D., Stace R., Ekneligoda T. 2017. Effect of high temperatures on sandstone – a computed tomography scan study. International Journal of Physical Modelling in Geotechnics 17(2):75–90.

McCabe S., Smith B. J., Warke P. A. 2007. Sandstone response to salt weathering following simulated fire damage: a comparison of the effects of furnance heating and fire. Earth Surface Processes and Landforms 32:1874–1883.

Pohle F., Jäger W. 2003. Material properties of historical masonry of the Frauenkirche and the masonry guideline for reconstruction. Construction and Building Materials 17:651–667.

Smith A. G., Pells P. J. N. 2008. Impact of fire on tunnels in Hawkesbury sandstone. Tunnelling and Underground Space Technology 23:65–74.

95

EXPLORING MICROBIAL COMMUNITIES INHABITING GYPSUM CRUSTS OF WEATHERED NATURAL BUILDING STONES

Laurenz Schröer^{1, 2}, Tim De Kock^{1, 3}, Nico Boon², Veerle Cnudde^{1, 4}

IN: SIEGESMUND, S. & MIDDENDORF, B. (EDS.): MONUMENT FUTURE: DECAY AND CONSERVATION OF STONE

– PROCEEDINGS OF THE 14TH INTERNATIONAL CONGRESS ON THE DETERIORATION AND CONSERVATION OF STONE –

VOLUME I AND VOLUME II. MITTELDEUTSCHER VERLAG 2020.

1 Ghent University, PProGRess-UGCT, Department of Geology, Ghent, Belgium

2 Ghent University, Center for Microbial Ecology and Technology (CMET), Ghent, Belgium

3 Antwerp Cultural Heritage Sciences (ARCHES), University of Antwerp, Antwerp, Belgium

4 Environmental hydrogeology, Utrecht University, Department of Earth Sciences, Utrecht, The Netherlands

Abstract

Microbes thrive in almost every possible environment, including natural building stones. Microbial communities affect these materials which can lead to biodeterioration. Among others, air pollution, especially SO₂ and NO_x, is an important actor for stone degradation. This leads to crust formation and in limestone typically to gypsum crusts. There are questions if and how sulphur-oxidizing prokaryotes can play a role in crust formation, oxidizing sulphur dioxide to sulphuric acid. This study explores the microbial community inside and underneath gypsum crusts to find out who is there and how they relate to the weathering and gypsum crust formation.

We studied Lede stone, a sandy limestone or calcareous sandstone, used in many historical buildings in north western Belgium. This stone is prone to weathering and gypsum crust formation. Two historic monuments have been sampled both in the urban environment (City hall, Ghent, Belgium) and in the countryside (Castle of Berlare, Belgium). These monuments consisted of severely weathered Lede stone: the City Hall in Ghent contained very thick botryoidal gypsum crusts while the crusts in Berlare were more superficial. Stone material was collected with a flame sterilized chisel and drill chuck and was used to isolate bacteria. To desribe the prokaryotic community: DNA was extracted, 16S rRNA genes were amplified and sequenced by Illumina Mi-Seq Next Generation Sequencing (NGS). The isolation campaign gave more information on the genus and species level of the bacterial community and makes it possible to test their abilities. The isolates from the two localities differ significantly and include diverse pigmented bacteria (orange, red, pink, yellow). The pigmented bacteria might contribute to the overall rock discoloration. The impact of the prokaryotic colonization on potential crust formation will be discussed.

Introduction

Our historic built heritage consists mainly out of natural building stones. As these stones interact with the environment, their properties slowly alter and the stones degrade. On limestones, gypsum crusts are among the most abundant deterioration features. These are sulphate encrustations that incorporate airborne dust, giving them a black appearance (Camuffo, Del Monte and Sabbioni, 1983). There is a strong correlation with pollution, especially with SO₂ and NO_x. It has been regarded that atmospheric SO₂ oxidizes and forms H₂SO₄, 96which will transform CaCO₃ to gypsum. NO_x acts as a catalyst (Bai, Thompson and Martinez-Ramirez, 2006).

Besides air pollution, biodeterioration by lichens, algae, fungi, archaea and bacteria can alter building stones significantly. Several groups of prokaryotes produce acids, chelating agents and pigments leading to dissolution and discolouration. (Doehne and Price, 2010). Some autotrophic prokaryotes can oxidize sulphur or nitrogen compounds and produce respectively H₂SO₄ or HNO₃. By other means they can turn air pollutants such as NO_x and SO₂ into nitrates and sulphates and can play a role in gypsum crust formation (Doehne and Price, 2010). Those prokaryotes have been isolated and sequenced from buildings and a correlation between air pollution and their occurrence has been indicated (Mansch and Bock, 1998; Villa et al., 2015; Li et al., 2016) Laboratory tests by Mansch and Bock (1996) show an increase of gypsum formation in the presence of nitrifying bacteria. The role of nitrogen and sulphur oxidizers in gypsum crust formation is still unclear and for this reason we sampled specifically gypsum crusts of Lede stone at the city and the countryside (De Kock et al., 2015), to characterize the prokaryotic community and to test if they can alter natural building stones.

This manuscript will focus on sulphur and nitrogen oxidizing prokaryotes and the discolouration potential of one of the isolates. A more in-depth description of the whole microbial community can be found (Schröer et al., 2020).

Material and Methods

The samples were retrieved at the beginning of April 2019 from two monuments in north western Belgium: six samples (G1–G6) from the City Hall of Ghent and seven (B1–B7) from the Castle of Berlare, representing respectively an urban and rural environment. Data from the Flemish environmental agency (VMM) indicates both a higher concentration of NO_x and SO₂ in the city centre of Ghent, compared to the area of Berlare. However, overall SO₂ concentrations declined around 90 % since the eighties resulting today in a minor difference between city and countryside. NO₂ emission decreased significantly as well, but here a bigger difference remains between urban (+− 30 μg/m³) and rural environment (+− 15 μg/m³) (Vlaamse Milieumaatschappij, 2019). Both monuments contain deteriorated Lede stone with gypsum crusts. Lede stone is a sandy limestone from north western Belgium out of the Lutetian (Eocene) (De Kock et al., 2015). The material for amplicon sequencing has been collected using a small flame sterilized drill, while around the drill hole, crust and underlying rock has been collected with a flame sterilised chisel to perform the isolations and soluble salt measurements.

Figure 1: A) Gypsum crust in Ghent and B) Berlare.

DNA was extracted out of the drill powder using the DNeasy PowerSoil Kit (Qiagen, Venlo, Netherlands), following the manufactures instructions. DNA extract was sent out to LGC genomics GmbH (Berlin, Germany) for 16S rRNA gene sequencing on an Illumina MiSeq platform and library preparation. For the bacteria it followed the same procedure as De Paepe et al. (2017) with 35 PCR cycles. Additionally, the archaea were determined, on three samples of each location, using a nested approach (De Vrieze et al., 2018).

Furthermore, one powdered sample of each location was used as inoculum for isolations: R2A agar for heterotrophic bacteria and thiosulphate plates for sulphur oxidizers containing (per Litre) 980 mL fresh water basal mineral medium, 9.7 g Na₂SO₄, 6 g Na₂S₂O_3, 10 g agar, 0.02 g bromothymol blue, 10 mL 971 M MOPS buffer, 10 mL 1 M NaHCO₃, 1 mL SL-10 trace element solution and 1 mL 7-vitamin solution The plates were incubated between two and six weeks at room temperature. For anaerobic isolations 2 g/L NaNO₃ was added. The isolates have been characterized by Sanger sequencing (LGC Genomics GMbH, Berlin, Germany) using 27 F and 1492R LGC primers. The resulting sequences were blasted with NCBI’s BLAST and with RDP Seqmatch. The gypsum crusts were characterized by their soluble ions. These have been extracted from powdered crust and underlying rock with Milli-Q water with a 1 : 5 ratio. The cations Na⁺, K⁺, Ca²⁺, Mg²⁺ and the anions Cl^–, NO₃^–, NO₂^–, SO₄^2–, PO₄^3– have been quantified on a 930 Compact Ion Chromatograph Flex (Methrohm, Switzerland) with a conductivity detector. From the measured concentrations, the amount of soluble ions was calculated. RUNSALT was used to model the phases of the salt mixture in function of relative humidity (Price, 2000; Bionda, 2005).

The potential discolouration of Arthrobacter agilis, one of the isolates was tested on the French oolitic Savonnières limestone. This stone is extensively used as a replacement for Lede stone (Dewanckele et al., 2014) This was tested by a water run-off test with a similar setup as De Muynck et al. (2009). Arthrobacter agilis was grown in R2A broth at 20 °C, after which it was sprinkled during two hours over six slaps of Savonnières limestone and dried during 24 hours. This was repeated two times more and in between its colour at two spots on the rock was examined with a point measurement using the CM-2600d spectrophotometer.

Results and Discussion

Gypsum crusts were present both in the urban and rural environment. These were thick botryoidal and black in Ghent, while in Berlare those were thinner and laminar with a rusty colour. The occurrence of the gypsum crusts has also been confirmed by the soluble salt analysis (see Table 1 for the anions). In every sample, there was a high concentration

of soluble SO₄^2– ranging between 1 mg/g_rock and 10 mg/g_rock. Although highly variable between the samples, overall, the soluble salt content was higher in the City Hall of Ghent compared to the samples of Berlare. This was also the case for the Cl^– and NO₃^–. Sample B6 was the only sample of Berlare with more Cl^– and NO₃^–, because of its sheltered position underneath a windowsill. Nitrite and phosphate were not found or only available in a very low amount. The compatible cations were primarily calcium (0.6−5 mg/g_rock) and in a lesser extend also potassium and magnesium. Sodium was most of the times slightly present, but could reach a high concentration (about 5 mg/g_rock for G4) combined with a high amount of soluble chloride. Based on the modelling performed using RUNSALT the accompanying salts (except gypsum), for the samples with high soluble salts content, at 20 °C and low RH were among others carnallite (KCl·MgCl₂.6H₂O), niter (KNO₃), nitromagnesite, (Mg(NO₃)₂.6H₂O), halite (NaCl), nitratine (NaNO₃) and Ca(NO₃)₂. These salts would be in solution at a relative humidity of about 60 % and more. For the other samples, the result of the model was more complex.

Within and underneath the gypsum crusts, 16S rRNA gene sequencing successfully identified the prokaryotic community on the City hall of Ghent and the Castle of Berlare, except for one sample (G1). Sulphur oxidizing, sulphur reducing and nitrogen oxidizers have been identified in some of the samples. Genera of nitrifying bacteria from different taxa were present: Nitrolancea (0.03 % 98in G2), Nitrospira (0.06 % in G2), Nitrosomonadaceae with Nitrosospira (0.002 % in G4, 0.7 % G6 and 0.0003 % in B1) and Nitrosomonas (0.002 % in G6). Archaea have been found in only one sample (G6) belonging to the nitrifying Nitrososphaeraceae.

Table 1: Soluble anions of the different samples in the urban and rural environment (mg/grock).

Some sulphur oxidizing bacteria were found as well, belonging to the “purple non-sulphur” bacteria (PNS). Despite their name, members of this group can oxidize low concentrations of sulphur (Hunter et al., 2009). PNS were abundant in G5 with 16 % of Rhodoplanes. Furthermore, there might be more PNS present in the samples as the order Rhodospirillales has been detected in G4 and G6 with an unclassified chemolithoautotrophic Magnetospira (0.2 % in G4) and with an unclassified genus of the family Rhodospirillaceae (0.3 % in G4). Furthermore, potential PNS in Rhodobacteraceae were represented as well, however again unclassified (0.5 % in G2, 0.002 % in G4, 0.4 % in G6 and 0.0003 % in B1) (Hunter et al., 2009; Williams et al., 2012). Beside sulphur oxidation, some identified bacteria belonged to the sulphur/sulphate reducing bacteria such as Desulfuromonadales (0.005 % in G3) and 4 % Geoalkalibacter in G5, but also Desulfobacteraceae, with Desulfofrigus (0.008 % in G5 and 0.0006 % in B4), Desulforhopalus (0.0006 % in B4) and unclassified genera (0.0009 % in G6, 0.003 % in B4 and 0.003 % in B6) (Greene, Patel and Yacob, 2009; Rosenberg et al., 2013).

There is a strong link between the location and the occurrence of the nitrogen oxidizing and sulphur bacteria. Both are present in selected samples across both locations, but mainly in the City Hall of Ghent, where in one sample they even reach high abundance. There is also a link between the occurrence of the nitrifying and sulphur prokaryotes and the higher soluble salt content of respectively nitrate or sulphate. However, also here there are some exceptions. The occurrence of sulphate reducing bacteria followed the same trend. These bacteria can cause further deterioration (Krumbein and Gorbushina, 2009) but besides inducing gypsum crust formation, they can reduce the sulphate and precipitate calcium carbonate. This process also produces sulphur and H₂S (Castanier, Le Métayer-Levrel and Perthuisot, 1999). As one sample in the city (G5) contains both a high concentration of PNS and sulphate reducing bacteria, it is not impossible that both groups are strongly linked together.

These results combine some of the findings of previous research by Mansch and Bock (1998) & Villa et al. (2015). The former found more nitrifying bacteria in the urban environment and the latter more sulphur oxidizing bacteria. It also confirms the findings of Li et al. (2016). However, members of sulphur compound oxidizing genera of which it is known that they deteriorate building stones, such as Thiobacillus (and related genera) have not been found (Krumbein and Gorbushina, 2009). This contrasts with the earlier findings of Villa et al. (2015). Furthermore, it is unclear if the PNS bacteria would affect natural building stones the same way as Thiobacillus. The presence of sulphur bacteria that only tolerate lower amounts of sulphur can be related to the decreasing SO₂ concentrations in the atmosphere. The low abundance of sulphur and nitrogen oxidizers, combined with the absence of those groups in several samples, indicates that other chemical factors are most likely still dominating gypsum crust formation. Although those gypsum crust have been formed since decades, when the SO₂ and NO_x concentrations were higher, it cannot be excluded that back then more sulphur and nitrogen oxidizers were present affecting crust formation.

Besides the metagenomic approach, also an isolation campaign has been performed. This did not succeed to retrieve chemolithoautotrophic prokaryotes. However, 20 genera belonging to 31 species of heterotrophic bacteria were successfully isolated and identified. Growth occurred mainly aerobic and denitrification occurred barely. Many isolates contained a red, orange, pink or yellow pigment and one of them, Arthrobacter agilis, has been successfully applied on the water run-off test (Figure 2). After one cycle of two hours, the rocks became significantly red and this has been confirmed by the spectrophotometric data. There was a dip between 450 and 560 nm in the reflectance, leading to an increase visibility of the complementary 99red/orange colours. During this test, we applied bacterial concentrations that do not occur in nature. However, it shows how easy it can discolour a natural building stone and the potential of some of the bacteria to cause aesthetic changes. Besides natural oxidation of iron-bearing minerals, these bacteria could also contribute to the rusty/red colour on the Castle of Berlare.

Figure 2: A) Spectral data showing the initial state of the Savonnières limestone (blank) and the progress of the colour change after adding Arthrobacter agilis during the different cycles (Cycle 1–3). B) Savonnières stone after three cycles showing the discolouration compared to the initial state at the top section.