International Biodeterioration & Biodegradation 53 (2004) 79–88 www.elsevier.com/locate/ibiod Identiÿcation of bacterial cultures from archaeological wood using molecular biological techniques Anne Christine Helmsa, Adam Camillo Martinyb, Jacob Hofman-Bangb, b b; Birgitte K. Ahring , Mogens Kilstrup ∗ aThe National Museum of Denmark, Department of Conservation, Brede, DK 2800 Lyngby, Denmark bBioCentrum-DTU, Section of Environmental Microbiology and Biotechnology, DK 2800 Lyngby, Denmark Received 22 May 2003; received in revised form 13 October 2003; accepted 20 October 2003 Abstract Anaerobic bacteria were isolated from a 1700-year-old wooden spear shaft, excavated from an archaeological site that dates from the iron age, in the southern part of Jutland, Denmark. The bacteria were cultivated in glucose- and xylose-supplemented media at 14◦C and 20◦C. A gene library with 21 clones was constructed by extracting and amplifying 16S rDNA sequences from the individual cultures. One clone was phylogenetically a"liated to the Spirochaeta. Eleven clones a"liated to an unidentiÿed member of the !-Proteobacteria were present in all culture samples. Three clones were a"liated to the ÿ-Proteobacteria. Four clones were clustered among the Geobacteriaceae, in the #-Proteobacteria. A single clone was clustered with gram-positives. All the identiÿed bacterial families are commonly found in soil or bog environments and many are able to utilize cellulose as their carbon or energy source. ? 2003 Elsevier Ltd. All rights reserved. Keywords: rDNA; Phylogeny; DNA 1. Introduction into factors which in#uence wood biodegradation (Gregory, 1998; Gregory et al., 2002; Hogan et al., 2001; Jordan, 2001; Archaeological wood may survive for thousands of years Powell et al., 2001). despite the numerous threats of both human and natural ori- One archaeological site that has received signiÿcant at- gin (Florian, 1990). The degree of preservation of buried tention in Denmark is Nydam Mose, which is an iron age wooden artefacts depends upon the nature of the environ- o$ering bog in the southern part of Jutland. It is a peat bog, ment at the archaeological site. Cold, dark and near anaer- where ground water has major in#uence on the conditions obic conditions result in slower degradation (Blanchette, in the bog. The peat is weakly acidic, i.e. pH 6–7 (Aaby 2000; Kim et al., 1996; Powell et al., 2001). With the et al., 1999). ever-increasing discovery of archaeological sites, but lack of At least ÿve or six di$erent depositions of war o$erings funds for excavation and conservation, it is becoming com- were made during the iron age. Analyses of the bog have mon to consider either in situ preservation or reburial of the shown that the ÿrst of these o$erings, which was around archaeological objects (Bergstrand, 2002; Brunning et al., 250 AD, was made in a fresh water lake. The youngest ÿnds 2000; Corÿeld, 2001; Gregory et al., 2002; Mouzouras et al., are dated at 475 AD. At that time, the lake had grown into 1990; Olsson, 2001; Pournou et al., 1998). In such cases, a swampy bog in which the weapons were plunged (Rieck it is necessary to ensure that further degradation is mini- et al., 1999). mized, in order to preserve our cultural heritage for future Excavations have been carried out in Nydam Mose since generations. Active monitoring of environmental parame- 1859. From 1992 to 1997, more than 15 000 mainly wooden ters is being undertaken world wide to gain further insight objects were uncovered. There seems to be at least an equal number of artefacts still left in the bog. Since 1996, a preservation project referred to as the in situ project, around ∗ Corresponding author. Tel.: +45-45-25-25-28; fax: +45-45-93-28-09. Nydam Mose has given a wealth of information. Today we E-mail address: [email protected] (M. Kilstrup). know the facts about oxygen content at di$erent ground 0964-8305/$ - see front matter ? 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2003.10.003 80 A. Christine Helms et al. / International Biodeterioration & Biodegradation 53 (2004) 79–88 levels, the peat acidity, the groundwater level and its tem- AD) in Nydam Mose in Denmark (UTM co-ordinates are perature, the dissolved ions and the redox and corrosion po- 546620/6090831 zone 32). This was one of many artefacts tentials (Gregory et al., 2002). Archaeological objects, both uncovered at a trial excavation in May 1999. wooden and metallic, from this site have been examined Immediately following excavation, all wooden artefacts extensively Gregory, 2001). The present study concerns a were cleaned on site, packed in perforated polyoleÿn ÿlm piece of a spear shaft, which was one of the 400 wooden and kept moist by covering with wet cotton textiles (Jensen objects excavated (Aaby et al., 1999). A subset of the col- et al., 1996). During the excavation period, the objects were lected objects, which consisted mainly of spear shafts and kept in closed plastic boxes in tap water. After about 2 arrows, was speciÿcally stored for research purposes, and weeks, the boxes were transported to the conservation work- the rest were conserved. shop and were stored together, still sealed, submerged in a The wooden objects from Nydam Mose appear visually 5000-l metal tank #ushed with running water. The sample to be intact immediately after excavation. However, when used for analysis was kept at the conservation workshop for the objects were allowed to dry, they collapsed. It has been about 14 months under these conditions. shown that the cellulose content in most of the artefacts is close to zero and that the density is only around 10% of 2.2. Methods and sample preparation its original value (Jensen, personal communication). The remaining material is composed almost exclusively of lignin. 2.2.1. Scanning electron microscopy of wooden objects In the case of ash wood, the density of the archaeological Photomicrographs of the physical appearance of the ar- 3 objects was around 0:1 g=cm . chaeological wooden sample was, together with those of a It is widely accepted, that bacteria are the main degraders fresh ash wood sample, were taken on a JSM-5310 LV scan- of archaeological wood under near-anaerobic conditions ning electron microscope. (Bjordal% et al., 1999, 2000; Blanchette and Ho$mann, 1994; Blanchette, 2000; Daniel and Nilsson, 1997; Gregory et al., 2.2.2. Growth conditions 2002; Kim et al., 1996; Kim and Singh, 2000; Powell The wooden sample (app. 5 1:4 cm2) was equilibrated et al., 2001). As it has not been possible to isolate these bac- in an anaerobic glove-box for about× 14 days. After anaero- teria by conventional methods (Bjordal% and Nilsson, 2002; bic equilibration the sample was trimmed and wood taken Kim and Singh, 2000; Nilsson and Daniel, 1983; Schmidt from the inner part was transferred to four 20-ml anaero- and Liese, 1994), the identiÿcation of bacteria that degrade bic culture #asks containing minimal BA-medium (DSMZ wood under such conditions has been based mainly on the medium no. 671, http://www.dsmz.de/media/med671.htm) micromorphology of the decay pattern (Daniel and Nilsson, without contaminating it with material from the surface of 1986; Daniel and Nilsson, 1997; Eaton and Hale, 1993; the wooden piece. Two of the anaerobic #asks were supple- Schmidt and Liese, 1994; Singh et al., 1990; Singh and mented with 0.2% (w/v) glucose and two with 0.2% (w/v) Butcher, 1991). The scope of the present work is to iden- xylose. The oxygen indicator dye resazurin was added to tify the microorganisms inhabiting archaeological wood in provide a visual indication of anaerobicity. The #asks were order to better understand the process of its degradation. incubated for 14 days at 14 C and 20 C. Controls without Knowledge of nutritional requirements of these microorgan- ◦ ◦ wood were incubated under the same conditions to exclude isms and the e$ect of environmental factors on degradation false positive results. could be used to model the degradation process as well as After incubation, the cells were re-inoculated by trans- to identify optimal sediment or soil types for reburial sites. ferring 100 l cells to 10 ml of fresh medium solution with To our knowledge, this paper describes the ÿrst success- ! the same carbon sources as mentioned above, and were in- ful cultivation of bacteria from the interior of archaeological cubated under the same conditions. wood. To allow cultivation of both anaerobic and faculta- tive anaerobic bacteria, we have used strict anaerobic growth conditions and DNA-based identiÿcation of these bacteria. 2.2.3. Microscopy By extracting DNA from the cultures, followed by ampliÿ- A drop of each culture was transferred to a microscope cation of the 16S rDNA regions, and construction of clone slide and examined by phase-contrast microscopy using a Carl Zeiss Axioplan epi#uorescence microscope ( 630). libraries, six di$erent bacteria have been identiÿed and their × full nucleotide sequences of the 16S rDNA regions have Digital images were captured with a 12-bit cooled slow-scan been determined and grouped phylogenetically. charge-coupled device camera. 2. Materials and methods 2.2.4. DNA extraction and puriÿcation Aliquots (500 !l) of each of the four cell suspensions di- 2.1. Material luted 10 fold were mixed with Proteinase K and cells were disrupted in a Fast Prep FP120 Savant Bead Beater. Chloro- The material for the present study was a piece of a form extraction of genomic DNA was performed according spear shaft from an iron age war o$ering (around 300 to Sambrook (Sambrook et al., 1989). The extracted DNA A. Christine Helms et al. / International Biodeterioration & Biodegradation 53 (2004) 79–88 81 was puriÿed twice with a Wizard DNA-clean-up system kit primers T3 (5"-AAT TTA CCC TCA CTA AAAG G-3") (WizardJ DNA-clean-up system, Promega) following the and T7 (5"-GTA ATA CGA CTC ACT ATA GG-3").