Applying high-throughput rRNA gene sequencing to assess microbial contamination of a 40-year old exposed archaeological profile

In recent years there has been a surge in the recovery of ancient organic molecules from archaeological contexts. These analyses are yielding unprecedented insights into human evolution and cultural practices, and are providing valuable data for reconstructing paleoenvironments. However, contamination of archaeological sediments by microorganisms can alter ancient biomolecular data. Furthermore, the extent to which microbes can penetrate ancient archaeological sediments once these are exposed by excavation is unknown. We tested this question at Crvena Stijena, a rock shelter in the Dinaric alps in Montenegro that contains archaeological deposits spanning more than 80,000 years. Excavations in the early 1960s exposed these profiles, which have been cleaned several times to permit sampling for archaeological, geological, and biomolecular analyses. The growth of green biofilms on the exposed profiles after cleaning has prompted the question of whether this surface contamination extends into the profile. To test this question, we examined five different geological layers by sampling sediments from the exposed surface and at 1 cm intervals horizontally into the profile. Results from 16S rRNA gene sequencing show that samples from sediment surfaces have distinct microbial communities from most samples collected more than 1 cm deep, and microbial biomass from the deeper samples is very low. Together, this evidence strongly indicates that microbial contamination is limited to the profile surfaces. This lowers the likelihood that ancient biomolecules in these sediments have been altered by recent changes to the in situ microbial community, and that cleaning of the profiles before sampling may not need to exceed 2 cm in depth. These results lend further support to the research utility of limited vertical sampling along archaeological profiles and witness sections, a strategy which conserves rare and limited archaeological deposits while helping to tackle key questions about the past.