Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging toChloroflexiSubphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

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Abstract
We previously reported that the thermophilic filamentous anaerobeAnaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylumChloroflexi, 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°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 ofAnaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties ofChloroflexisubphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze theChloroflexisubphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targetedChloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with theChloroflexisubphylum 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 aChloroflexi-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 theChloroflexisubphylum 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 theChloroflexisubphylum 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.

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