Granular sludge processes are frequently used in domestic and industrial wastewater treatment. The granule buoyant density and biomass density are important parameters for the design and operation of granular sludge reactors. Different methods to measure the granule density include the pycnometer method, the Percoll density gradient method, the dextran blue method, and the settling velocity method. In this study, a comparison was made between these four methods to measure granule density for granules from a full-scale granular sludge plant treating domestic sewage. The effect of salinity on granule density was assessed as well. Three out of the four evaluated methods yielded comparable results, with granule buoyant densities between 1025.7 and 1028.1 kg/m³ and granule biomass densities between 71.1 and 71.5 g/L (based on volatile suspended solids (VSS)). The settling velocity method clearly underestimated the granule density, due to the complex relation between granule properties and settling velocity. The pycnometer method was the most precise method, but it was also quite susceptible to bias. The granule buoyant density increased proportionally with salinity, to 1049.2 kg/m³ at 36 g/L salinity. However, the granule biomass density, based on VSS, remained constant. This showed that the granule volume was not affected by salinity and that the buoyant density increase was the result of diffusion of salts into the granule pores. Overall, the granule density can be measured reliably with most methods, as long as the effect of salinity is considered. The results are discussed in light of operational aspects for full-scale granular sludge plants.

In aerobic granular sludge (AGS) reactors, granules of different sizes coexist in a single reactor. Their differences in settling behaviour cause stratification in the settled granule bed. In combination with substrate concentration gradients over the reactor height during the anaerobic plug-flow feeding regime, this can result in functional differences between granule sizes. In this study, we compared the hydrolytic activity in granules of 4 size ranges (between 0.5 and 4.8 mm diameter) collected from a full-scale AGS installation. Protease and amylase activities were quantified through fluorescent activity assays. To visualise where the hydrolytic active zones were located within the granules, the hydrolysis sites were visualized microscopically after incubating intact and sliced granules with fluorescent casein and starch. The microbial community was studied using fluorescent in situ hybridization (FISH) and sequencing. The results of these assays indicated that hydrolytic capacity was present throughout the granules, but the hydrolysis of bulk substrates was restricted to the outer 100 µm, approximately. Many of the microorganisms studied by FISH, such as polyphosphate and glycogen accumulating organisms (PAO and GAO), were abundant in the vicinity of the hydrolytically active sites. The biomass-specific hydrolysis rate depended mainly on the available granule surface area, suggesting that different sized granules are not differentiated in terms of hydrolytic capacity. Thus, the substrate concentration gradients that are present during the anaerobic feeding in AGS reactors do not seem to affect hydrolytic activity at the granule surfaces. In this paper, we discuss the possible reasons for this and reflect about the implications for AGS technology.

Biofilm and granular sludge processes depend on diffusion of substrates. Despite their importance for the kinetic description of biofilm reactors, biofilm diffusion coefficients reported in literature vary greatly. The aim of this simulation study was to determine to what extent the methods that are used to measure diffusion coefficients contribute to the reported variability. Granular sludge was used as a case study. Six common methods were selected, based on mass balances and microelectrodes. A Monte Carlo simulation was carried out to determine the theoretical precision of each method, considering the uncertainty of various experimental parameters. A model-based simulation of a diffusion experiment was used to determine the theoretical accuracy as a result of six sources of error: solute sorption, biomass deactivation, mass transfer boundary layer, granule roughness, granule shape, and granule size distribution. Based on the Monte Carlo analysis, the relative standard deviation of the different methods ranged from 5% to 61%. In a theoretical experiment, the six error sources led to an 37% underestimation of the diffusion coefficient. This highlights that diffusion coefficients cannot be determined accurately with existing experimental methods. At the same time, the need for measuring precise diffusion coefficients as input value for biofilm modeling can be questioned, since the output of biofilm models has a limited sensitivity toward the diffusion coefficient.