Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million -gal-per-day industrial wastewater treatment plant was investigated using r RNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sl udge samples taken on different dates. Partial rRNA gene sequences were obt ained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtai ned for 18 clones. Seventeen of these clones were members of the beta subdi vision, and their sequences showed high homology to sequences of known bact erial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of whic h showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolate d from this treatment plant. A nearly full-length 16S rDNA sequence was obt ained from Hyphomicrobium sp, strain M3. Phylogenetic analysis revealed tha t Hyphomicrobium sp, strain M3 was 99% similar to Hyphomicrobium denitrific ans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobiu m cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organis ms (95 to 97% similarity). whole-cell fluorescence hybridization of microor ganisms in the activated sludge with genus-specific Hyphomicrobium probe S- G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and reveal ed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the flee structure. Dot blot hybridization of activated sludge s amples from 1995 with probes designed for Hyphomicrobium cluster I and Hyph omicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12 -fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA i n the activated sludge.