In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I

Abstract

We previously showed that very thin filamentous bacteria affiliated with the division green non-sulfur bacteria were abundant in the outermost layer of thermophilic methanogenic sludge granules fed with sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol. 65:1280-1288, 1999). Further 16S ribosomal DNA (rDNA) cloning-based analysis revealed that the microbes were classified within a unique clade, green non-sulfur bacteria (GNSB) subdivision I, which contains a number of 16S rDNA clone sequences from various environmental samples but no cultured representatives. To investigate their function in the community and physiological traits, we attempted to isolate the yet-to-be-cultured microbes from the original granular sludge. The first attempt at isolation from the granules was, however, not successful. In the other thermophilic reactor that had been treating fried soybean curd-manufacturing wastewater, we found filamentous microorganisms to outgrow, resulting in the formation of projection-like structures on the surface of granules, making the granules look like sea urchins. 16S rDNA-cloning analysis combined with fluorescent in situ hybridization revealed that the projections were comprised of the uncultured filamentous cells affiliated with the GNSB subdivision I and Methanothermobacter-like cells and the very ends of the projections were comprised solely of the filamentous cells. By using the tip of the projection as the inoculum for primary enrichment, a thermophilic, strictly anaerobic, filamentous bacterium, designated strain UNI-1, was successfully isolated with a medium supplemented with sucrose and yeast extract. The strain was a very slow growing bacterium which is capable of utilizing only a limited range of carbohydrates in the presence of yeast extract and produced hydrogen from these substrates. The growth was found to be significantly stimulated when the strain was cocultured with a hydrogen-utilizing methanogen, Methanothermobacter thermautotrophicus, suggesting that the strain is a sugar-fermenting bacterium, the growth of which is dependent on hydrogen consumers in the granules.

FIG. 1

Phylogenetic tree of GNSB, including environmental clones. The 16S rDNA sequences obtained in the previous study (MUG and TUG clones) and reference sequences were aligned, and phylogenetic trees were constructed by the neighbor-joining method (pairwise analysis). Bar represents 5 nucleotide substitutions per 100 nucleotides in 16S rDNA sequences. The sequence of Aquifex pyrophilus was used to root the tree. Bootstrap values above 50% are indicated at the branch points. The accession number of each reference sequence and the origin of environmental clones are also shown in parentheses. Names of cultivated organisms are shown in bold and italic. Our UASB clones, referred to as thermophilic UASB cluster and strain UNI-1, are shown in a bracket.

FIG. 2

Photographs of granules formed in reactor II. (A) Sludge granules at day 91 of operation. (B) Fluffy granules formed at day 133. (C) Magnified view of a fluffy granule.

FIG. 3

Scanning electron micrographs of spine-like projections sticking out of the fluffy granules (sea urchin granules) in reactor II. (A) Whole view of a projection (bar, 150 μm). (B) Higher magnification of the bottom part of the projection (bar, 20 μm). (C) Higher magnification of the tip of the projection (bar, 30 μm)

FIG. 4

In situ hybridization of a projection from the fluffy thermophilic granules viewed by confocal laser scanning microscopy (digitally produced images with pseudocolors). The projection was simultaneously hybridized with Cy5-labeled archaeal probe ARC915 (indicated as red) and rhodamine-labeled probe GNSB633 for thermophilic UASB cluster of GNSB subdivision I (indicated as green). (A) Magnified view of the bottom part of the projection (bar, 50 μm). (B) Magnified view of the tip portion of the projection (bar, 50 μm).

FIG. 5

In situ hybridization of strain UNI-1 cells isolated in this study. The cells were hybridized with the rhodamine-labeled GNSB633 probe. (A) Phase-contrast micrograph of strain UNI-1 cells (bar, 10 μm). (B) Fluorescent micrograph of the same field as panel A, showing that all cells were stained with the thermophilic UASB cluster-specific probe GNSB633.

FIG. 6

Fermentation of glucose by strain UNI-1 in the presence (A) and the absence (B) of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain ΔH. The amounts of gases are shown as millimoles of gas produced per liter of culture.

FIG. 7

Alignment of target sequences of the EUB338 probe suite with the corresponding regions of 16S rRNA of members of the GNSB group, showing mismatches with the Bacteria universal probe EUB338 and its sequence variations (EUB338-II and EUB338-III). Mismatches with the EUB338 probe sequence are shaded.