Comparative evaluation of multiple methods to quantify and characterise granular anammox biomass

Six methodologically different approaches were evaluated and compared regarding their suitability to quantify and characterise granular anammox biomass. The investigated techniques were gravimetric analysis (GA), activity measurements (AM), Coulter counter analysis (CC), quantitative PCR (qPCR), heme protein quantification (HQ) and the novel image analysis technique Particle Tracking (PT). The focus was set on the development of fast, economic and user-friendly approaches for potential implementation in regular wastewater treatment plant (WWTP) monitoring. To test the effectiveness of each technique, two sample matrices were chosen at the WWTP Strass (Austria): i) sludge liquor of the DEMON tank, treating ammonium-rich reject water of anaerobic digestion via the deammonification process and rich in anammox biomass (SL), and ii) the mainstream biological stage, that has been enriched with anammox biomass for more than two years (B). In both of these plants hydro-cyclones are installed for density-fractioning of the sludge into a low- and a high-density fraction, thus leading to a characteristic anammox distribution in the investigated sample set. All investigated methods could statistically discriminate the SL samples. Heme quantification and qPCR were also able to correctly classify the B-samples and both methods showed a Pearson's correlation coefficient of 0.81. An asset of the PT and CC method is the additional qualitative characterization of granule size distribution that can help to better understand and optimise general process operation (cyclone operation duration and construction characteristics). In combination these two methods were able to elucidate the relationship of gross granule volume and actual biomass, excluding the dead volume of inner cavities and exopolymers. We found a linear sphere-equivalent-radius correction factor (3.96 ± 0.15) for investigated anammox granules, that can be used for the fast and reliable PT technique to avoid biomass overestimation. We also recommend routine HQ and PT analysis as ideal monitoring strategy for anammox abundance in wastewater facilities with the HQ technique entailing the further advantage of being also suited for non-granular anammox biomass.