Bacterial community in the intertidal sediments populated by Arenicola marina, a terminal restriction fragment length polymorphisms study

Abstract

Sediments offer microorganisms an unexplored numbers of niches with the opportunity to evolve specialized microbial communities. The small size of microbial niches in biogeochemical gradients in sediments called for a high resolution study of the populations. We applied a genetic fingerprint method, the terminal restriction fragment polymorphism (T-RFLP), to characterize the diversity of the 16S rRNA gene present in thin sediment layers at which one TRF represents one operational taxonomic unit (OTU). A partial gene amplification and restriction enzyme digestion of the amplicon allows the detection of about 150 different fragments in an intensity range of 100 to 10000 relative fluorescence units as a picture of the richness and evenness of the bacterial community.The T-RFLP method was established for intertidal soft sediments from the Wadden Sea, the North Sea. The variations in the results were correlated to variations in individual steps of the method protocol. Restriction enzyme digest and digest analysis on a capillary sequencer correlated with a dissimilarity of about 20% and 10% in the obtained replicate datasets describing one pooled amplicon from one DNA sample after binning with a fixed window size of 0.5 and 1 base pair, respectively. The biases in individual PCR reactions did not increase the dissimilarity after performing independent T-RFLP analyses from one DNA sample. The dissimilarity was partly caused by an imperfect binning. Working with a high resolution window size of 0.5 bp, no starting point (50.25, 50.20, 50.30 and 50.65 bp) gave a perfect binning result. Some of identical TRFs were always binned into two different TRFs, thus creating an additional OTU. A window size of 1 bp with starting point 50.50 bp gave similar dissimilarities. Although our results may require an improved binning technique to utilize the full biodiversity information in the profiles, the current T-RFLP technique clearly detected the biological variation in adjacent small sediment layers and can be used to characterize the microbial community in individual sediment layers. Eukaryotes offer and create a number of niches. The lugworm Arenicola marina is a bioturbator in marine intertidal sediments. The T-RFLP method was applied to investigate the bacterial community in the burrow of the lugworm A. marina. The U-shaped burrow is divided into three compartments: the vertical head shaft tube through which the surface sediment is sinking down and ingested by the lugworm, the horizontal gallery tube at where the lugworm relatively stays permanent inside the sediment and the vertical tail shaft tube through which the lugworm does defecation by moving backward until the tail reaches sediment surface and ejects characteristic fecal cast on the sediment surface. In the bulk sediment surrounding the U-shaped burrow, the sediment contained a number of different bacterial communities changing with depth. On the basis of an aerobic layer, a redox potential discontinuity (RDP) layer and an anoxic layer, the decreasing and the increasing TRFs over depth may represent surface and subsurface layer bacteria respectively at 0-2 cm and 2-10 cm depth. T-RFLP data suggested that the RDP layer is at 3-5 cm sediment depth, because the unique TRFs of the surface layer and subsurface layers were not found at this depth and the change of abundance of TRFs was fast. The T-RFLP analyses clearly grouped the microbial population in the head shaft tube with sediment surface populations. The tail shaft tube was populated by different populations; close to the surface dominated by the surface bacteria and below 3 cm dominated by the subsurface bacteria. The populations in the gallery tube were similar to those in the head and tail shaft tube. The richness in the gallery tube was the lowest but had the highest evenness. T-RFLP analyses of two mm-thick sediment layers from areas with A. marina and without A. marina also revealed a strong depth-dependence of the surface bacterial community composition. According to the T-RFLP analyses, the presence or absence of A. marina had no clear detectable influence on the microbial populations in the top two centimeter of sediment. Most likely, the increase presence of other burrowing animals in the A. marina exclusion areas seems to form highly similar biogeochemical environments for the development of bacterial communities.