Environmental Microbiology (2006) 8(4), 709–719 doi:10.1111/j.1462-2920.2005.00949.x © 2005 The Authors Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd sphere bacteria in tidal flatsR. Wilms et al. Received 22 July, 2005; accepted 30 September, 2005. *For corre- spondence. E-mail engelen@icbm.de; Tel. (+49) 441 798 5376; Fax (+49) 441 798 3583. Present address: † School of Earth, Ocean and Planetary Sciences, Cardiff Uni, Main Building, Park Place, Cardiff CF10 3YE, Wales, UK; ‡ Senckenberg Institute, Division of Marine Science, Südstrand 40, D-26382 Wilhelmshaven, Germany. Deep biosphere-related bacteria within the subsurface of tidal flat sediments Reinhard Wilms, Beate Köpke, Henrik Sass, † Tae Soo Chang, ‡ Heribert Cypionka and Bert Engelen* Institut für Chemie und Biologie des Meeres, Universität Oldenburg, Carl-von-Ossietzky Straße, 9-11, D-26129 Oldenburg, Germany. Summary Biogeochemical and microbiological processes in the upper sediment layers of tidal flats were analysed in many investigations, while deeper zones remained largely unexplored. Therefore, denaturant gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments along the depth profile of up to 5.5 m-long sediment cores was performed in comparison with lithological and geochemical parameters. The investi- gation revealed that different compartments of the sediment columns were characterized by specific microbial communities. These compartments were analysed by sequencing of 113 DGGE bands. The upper layers down to 160–200 cm were dominated by gamma- and delta- Proteobacteria representing more than 60% of the total number of phylotypes. Under- neath, a striking shift in community composition was observed, as the Proteobacteria were replaced by Chloroflexi with more than 60% of all sequences. As sulfate was still available as an electron acceptor in these layers, the abundance of Chloroflexi might be promoted by the electron donor or the quality of the carbon source. The dominance of this group, previ- ously known as green non-sulfur bacteria, indicates the presence of a typical deep-biosphere microbial community in relatively young subsurface sediments. Thus, tidal flats might offer a convenient possibility to study and understand certain aspects of the deep biosphere in general. Introduction One of the largest coherent tidal flat systems in the world is the Wadden Sea, a 500 km-long, shallow and nutrient- rich area along the southern North Sea coast between Den Helder (the Netherlands) and Esbjerg (Denmark). It arose after the last ice age about 7500 years ago with the generation of the first islands (Flemming, 1992). The Wad- den Sea is a dynamic system with a strong three-dimen- sional variability. Intense bioturbation occurs and reaches down to at least 20 cm depth (Kristensen, 2000), forming a patchy mosaic of physico-chemical and biological microenvironments. Additionally, tidal flats are subjected to sporadically occurring storm events that lead to resus- pension of large sediment patches making correct age determinations of different layers difficult to obtain. Fur- thermore, intercalations of peat, which are older than the Wadden Sea itself, interfere with the analysis of relation- ships between age and depth. Tidal dynamics lead to an intense exchange of organic and inorganic nutrients with the open sea but the Wadden Sea also receives a remarkable nutrient input from the adjacent mainland. As a result, the system is character- ized by a high productivity in the water column and the benthos (Dittman, 1999; Poremba et al ., 1999). Intense remineralization processes occur within the water column but mostly within the uppermost sediment layers where they were analysed intensely (Rasmussen and Jør- gensen, 1992; Jørgensen, 2000). So far, the analysis of sediment-dwelling microbial communities from tidal flats using culture-independent (Böttcher et al ., 2000; Llobet-Brossa et al ., 2002; Rütters et al ., 2002) or cultivation-based methods (Brinkhoff et al ., 1998; Llobet-Brossa et al ., 1998; Mußmann et al ., 2003) were restricted to the uppermost 50 cm. For instance, a study applying fluorescence in situ hybridization to anal- yse microbial community structures of the sediment sur- face down to 20 cm depth revealed the dominance of members of the Proteobacteria and Bacteriodetes (Llo- bet-Brossa et al ., 1998). Investigations on deeper coastal sediments are still rare and generally focused on bio- geochemical parameters (Chambers et al ., 2000). Thom- sen and colleagues (2001) demonstrated the presence of active sulfate-reducing and methanogenic communities in a 6 m-long sediment core from Aarhus Bay (Denmark). However, recent studies on subsurface habitats have shown a great variety of microbial life within much deeper and older marine sediments (Parkes et al ., 1994; Pedersen, 2000; Coolen et al ., 2002; D’Hondt et al ., 2004). The aim of our study was to give a first insight into the