Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

Abstract

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.

FIG. 1.

Evolutionary dendrogram of clones and isolates obtained from mesophilic and thermophilic sludge granules in the bacterial phylum Chloroflexi, based on 16S rRNA gene sequences. The sequences were aligned, and the phylogenetic tree was constructed by the neighbor-joining method. Bar = 5 nucleotide substitutions per 100 nucleotides. 16S rRNA gene sequences of Thermotogae were used to root the tree. Bootstrap values greater than 50% are indicated at branch points. The accession number of each reference sequence and the origins of the environmental clones are indicated in parentheses. Our clones and isolates used in this study are indicated by boldface type; the clones retrieved from mesophilic sludges are indicated by blue, the clones retrieved from thermophilic sludges are indicated by red, and strains isolated in this study are indicated by purple.

FIG. 2.

Fluorescence in situ hybridization of sections from three mesophilic sludge granules, viewed using confocal laser scanning microscopy. (A and D) AMG sludge granule; (B and E) BMG sludge granule; (C and F) CMG sludge granule. The sections in panels A to C were hybridized with the Cy-3-labeled EUB338 probe, specific for the domain Bacteria (green), and the Cy-5-labeled ARC915 probe, specific for the domain Archaea (red); and the sections in panels D to F were hybridized with the Cy-3-labeled GNSB941 probe, specific for the bacterial phylum Chloroflexi (green), and the Cy-5-labeled ARC915 probe, specific for the domain Archaea (red). Bars = 50 μm.

FIG. 3.

Scanning electron micrographs of whole sludge granules (A to C) and the surfaces of the sludge granules (D to F). (A and D) AMG sludge granule; (B and E) BMG sludge granule; (C and F) CMG sludge granule.

FIG. 4.

Micrographs of a floc found in the AMG reactor. (A) Phase-contrast and (B) fluorescence micrographs of the same field, showing that all filamentous cells in the floc hybridized with the Chloroflexi-specific probe GNSB941. Bar = 50 μm.

FIG. 5.

Phase-contrast micrographs showing the cell morphology of mesophilic and thermophilic isolates affiliated with Chloroflexi subphylum I. (A) Strain IMO-1. Bar = 10 μm. (B) Strain KIBI-1. Bar = 10 μm. (C) Strain YMTK-2. Bar = 20 μm.