Diagnosis and quantification of glycerol assimilating denitrifying bacteria in an integrated fixed-film activated sludge reactor via 13C DNA stable-isotope probing

Glycerol, a byproduct of biodiesel and oleo-chemical manufacturing operations, represents an attractive alternate to methanol as a carbon and electron donor for enhanced denitrification. However, unlike methanol, little is known about the diversity and activity of glycerol assimilating bacteria in activated sludge. In this study, the microbial ecology of glycerol assimilating denitrifying bacteria in a sequencing batch integrated fixed film activated sludge (SB-IFAS) reactor was investigated using (13)C-DNA stable isotope probing (SIP). During steady state SB-IFAS reactor operation, near complete nitrate removal (92.7 ± 5.8%) was achieved. Based on (13)C DNA clone libraries obtained after 360 days of SB-IFAS reactor operation, bacteria related to Comamonas spp. and Diaphorobacter spp. dominated in the suspended phase communities. (13)C assimilating members in the biofilm community were phylogenetically more diverse and were related to Comamonas spp., Bradyrhizobium spp., and Tessaracoccus spp. Possibly owing to greater substrate availability in the suspended phase, the glycerol-assimilating denitrifying populations (quantified by real-time PCR) were more abundant therein than in the biofilm phase. The biomass in the suspended phase also had a higher specific denitrification rate than the biofilm phase (p = 4.33e-4), and contributed to 69.7 ± 4.5% of the overall N-removal on a mass basis. The kinetics of glycerol based denitrification by suspended phase biomass were approximately 3 times higher than with methanol. Previously identified methanol assimilating denitrifying bacteria were not associated with glycerol assimilation, thereby suggesting limited cross-utilization of these two substrates for denitrification in the system tested.