Identification of glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal plant by stable isotope probing

Microbiology in wastewater treatment has mainly been focused on problem-causing filamentous bacteria or bacteria directly involved in nitrogen and phosphorus removal, and to a lesser degree on flanking groups, such as hydrolysing and fermenting bacteria. However, these groups constitute important suppliers of readily degradable substrates for the overall processes in the plant. This study aimed to identify glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plant (WWTP), and to determine their abundance in similar WWTPs. Glucose-fermenting micro-organisms were identified by an in situ approach using RNA-based stable isotope probing. Activated sludge was incubated anaerobically with ¹³C₆-labelled glucose, and ¹³C-enriched rRNA was subsequently reverse-transcribed and used to construct a 16S rRNA gene clone library. Phylogenetic analysis of the library revealed the presence of two major phylogenetic groups of Gram-positive bacteria affiliating with the genera Tetrasphaera, Propionicimonas (Actinobacteria), and Lactococcus and Streptococcus (Firmicutes). Specific oligonucleotide probes were designed for fluorescence in situ hybridization (FISH) to specifically target the glucose-fermenting bacteria identified in this study. The combination of FISH with microautoradiography confirmed that Tetrasphaera, Propionicimonas and Streptococcus were the dominant glucose fermenters. The probe-defined fermenters were quantified in 10 full-scale EBPR plants and averaged 39 % of the total biovolume. Tetrasphaera and Propionicimonas were the most abundant glucose fermenters (average 33 and 4 %, respectively), while Streptococcus and Lactococcus were present only in some WWTPs (average 1 and 0.4 %, respectively). Thus the population of actively metabolizing glucose fermenters seems to occupy a relatively large component of the total biovolume.