Niche Differentiation of Phenol-Degrading Microorganisms in UASB Granular Sludge as Revealed by Fluorescence in situ Hybridization

A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization (FISH) and clone library construction. Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation. For accurate taxonomic assignment of Cryptanaerobacter clones, phylogenetic analysis using nearly full-length 16S ribosomal RNA (rRNA) gene sequences was necessary. Three oligonucleotide probes were designed to detect the following three taxonomic groups: Syntrophorhabdaceae, Cryptanaerobacter, and Syntrophus. FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea. However, a well-defined distribution of Syntrophorhabdaceae, Cryptanaerobacter, and Syntrophus was observed. Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other, while Syntrophorhabdaceae was located in the deeper part of the granules. Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate. Phenol degradation in the granular sludge was observed to be carried out in the following way. First, Cryptanaerobacter converts phenol to benzoate, which is then degraded by Syntrophus into acetate. This syntrophic degradation of phenol occurs near the surface of the granule, where the phenol concentration is high. In the deeper part of the granule, where the phenol concentration is lower, Syntrophorhabdaceae degrades phenol into acetate. We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms, which contradicts the theories presented by previous studies.