Identification and ecophysiological characterization of epiphytic protein-hydrolyzing saprospiraceae ("Candidatus Epiflobacter" spp.) in activated sludge

Abstract

The identity and ecophysiology of a group of uncultured protein-hydrolyzing epiphytic rods attached to filamentous bacteria in activated sludge from nutrient removal plants were investigated by using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. The epiphytic group consists of three closely related clusters, each containing 11 to 16 clones. The closest related cultured isolate is the type strain Haliscomenobacter hydrossis (ATCC 27775) (<87% similarity) in the family Saprospiraceae of the phylum Bacteroidetes. Oligonucleotide probes at different hierarchical levels were designed for each cluster and used for ecophysiological studies. All three clusters behaved similarly in their physiology and were specialized in protein hydrolysis and used amino acids as energy and carbon sources. They were not involved in denitrification. No storage of polyphosphate and polyhydroxyalkanoates was found. They all colonized probe-defined filamentous bacteria belonging to the phyla Chloroflexi, Proteobacteria, and candidate phylum TM7, with the exception of cluster 1, which did not colonize TM7 filaments. The three epiphytic clusters were all widespread in domestic and industrial wastewater treatment plants with or without biological phosphorus removal, constituting, in total, up to 9% of the bacterial biovolume. A new genus, "Candidatus Epiflobacter," is proposed for this epiphytic group in activated-sludge treatment plants, where it presumably plays an important role in protein degradation.

FIG. 1.

(A) Simplified distance tree (neighbor joining) built for all of the genera and the main microbial groups in the family Saprospiraceae of the phylum Bacteroidetes. (B) Distance tree built for the three clusters of “Candidatus genus Epiflobacter” as shown in panel A. (C) Distance tree showing the Skagen clones targeted by probe Bac111. The sequences with bold names in panels A and B were obtained in this study. The values in the quadrangles of panel A represent the numbers of clones in the clone clusters. The bootstrap values in all of the panels (only those >50% are shown) were calculated on the basis of 1,000 resamplings. The scale bars in all panels represent 1 substitution per 10 nucleotides.

FIG. 2.

(A) FISH image of activated sludge after color combination showing Bacteria hybridized with EUBmix (Cy5 labeled, set as green) as cells labeled with different colors. Epiflora hybridized with Bac111 (FLUOS labeled, set as blue) as cyan-labeled cells (mixture of green and blue, indicated by arrows b), and epiflora hybridized with EpiMix (Cy3 labeled, set as red) as purple-labeled cells (mixture of blue, red, and green, indicated by arrows a). (B, C, and D) MAR-FISH image of activated sludge with epiflora. (B) FISH image of epiflora hybridized with EpiMix (Cy3 labeled, set as red) and Bacteria hybridized with EUBmix (FLUOS labeled, set as green). (C) Bright-field image of MAR of the same field as panel B. (D) Overlay of images D and C. Images B, C, and D show that the epiflora hybridized with EpiMix (e.g., those indicated by arrows) take up a mixture of labeled amino acids under aerobic conditions. The bars in panels A and B represent 10 μm.