DISTRIBUTION OF SULFATE-REDUCING BACTERIA, O2, AND H2S IN PHOTOSYNTHETIC BIOFILMS DETERMINED BY OLIGONUCLEOTIDE PROBES AND MICROELECTRODES

The vertical distribution of sulfate-reducing bacteria (SRB) in photos ynthetic biofilms from the trickling filter of a sewage treatment plan t was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen penetration, we incubated two 4-mm-thick biofilm samp les in darkness or exposed to light at natural intensity. Gradients of O2, H2S, and pH were examined with microelectrodes during incubation. The samples were subsequently frozen with liquid nitrogen and sliced on a cryomicrotome in 20-mum vertical slices. Fluorescent-dye-conjugat ed oligonucleotides were used as ''phylogenetic'' probes to identify s ingle cells in the slices. Oligonucleotide sequences were selected whi ch were complementary to short sequence elements (16 to 20 nucleotides ) within the 16S rRNA of sulfate-reducing bacteria. The probes were la beled with fluorescein or rhodamine derivatives for subsequent visuali zation by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta grou p of purple bacteria), Desulfobacter spp., and a nonhybridizing contro l. The SRB were unevenly distributed in the biofilm, being present in all states from single scattered cells to dense clusters of several th ousand cells. To quantify the vertical distribution of SRB, we counted cells along vertical transects through the biofilm. This was done in a blind experiment to ascertain the reliability of the staining. A neg ative correlation between the vertical distribution of positively stai ned SRB cells and the measured O2 profiles was found. The distribution differed in light- and dark-incubated samples presumably because of t he different extensions of the oxic surface layer. In both cases the S RB were largely restricted to anoxic layers.