Shearing of biofilms enables selective layer based microbial sampling and analysis

Granules are large, self-supporting biofilms that form naturally in high-rate anaerobic treatment systems and are extremely important to reactor functionality. Granules exhibit functional and phylogenetic layering, interesting to both scientists and technologists. Until now, it has only been possible to analyze layering through sectioning and microscopic analysis with fluorescent in situ hybridization, or to analyze the whole granule through DNA extraction and microbial community profiling methods. This means different functional and spatial layers cannot be analyzed separately, including next generation sequencing techniques, such as pyrotag sequencing. In this work, we describe a method to remove microbes selectively from successive spatial layers through hydraulic shearing and demonstrate its application on anaerobic granules of three different types (VFA-, carbohydrate-, protein-fed) in size ranges 0.6-2 mm. Outer layers in particular could be selectively sheared as confirmed by FISH. TRFLP was used as an example bulk DNA method on selectively sheared fractions. A shift in dominant population was found from presumptive acidogens (such as Bacteroidetes and Anaerolinea) in outer layers to syntrophs (such as Syntrophomonas and Geobacter) in inner layers, with progressive changes through the depth. The strength of the shear-bulk molecular method over FISH was that a deeper phylogenetic profile could be obtained, even with TRFLP, and that prior knowledge of the community is not required.