Читать книгу Monument Future - Siegfried Siegesmund - Страница 125

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.