Proteins and carbohydrates are both major biodegradable fractions in wastewater. Complexation with coexisting compounds, such as iron (Fe) and humic acids (HA), which are both commonly present in wastewater, could influence the different degradation rates of proteins and carbohydrates. Depending on the redox conditions, Fe exists as Fe(II) or Fe(III), with differing binding affinities and chemical behaviour. This research aims to systematically assess the complex interaction between Fe, protein, and HA compounds under aerobic and anaerobic conditions. The results showed that the addition of Fe(III) and HA to a protein solution inhibited its hydrolysis rate by more than 90 % under aerobic conditions. In contrast, interactions between the same compounds and carbohydrates were much weaker and had a minimal effect on hydrolysis rates. Complexation with Fe, proteins, and HA was indicated by increased molecular sizes and reduced concentrations of free iron, protein, and HA. FTIR results showed that Fe(III) formed complexes with proteins and HA through electrostatic and coordination bonds involving various functional groups. Anaerobic reduction of Fe(III) to Fe(II) by hydrazine resulted in weaker binding and the formation of smaller, less stable protein–humic acid complexes. These findings suggested that modulating Fe complexation under alternating aerobic and anaerobic conditions, such as those found in redox-cycling wastewater treatment, can be a promising strategy to enhance protein degradation.

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.

Despite aerobic granular sludge wastewater treatment plants operating around the world, our understanding of internal granule structure and its relation to treatment efficiency remains limited. This can be attributed in part to the drawbacks of time-consuming, labor-intensive, and invasive microscopy protocols which effectively restrict samples sizes and may introduce artefacts. Time-domain nuclear magnetic resonance (NMR) allows non-invasive measurements which describe internal structural features of opaque, complex materials like biofilms. NMR was used to image aerobic granules collected from five full-scale wastewater treatment plants in the Netherlands and United States, as well as laboratory granules and control beads. T1 and T2 relaxation-weighted images reveal heterogeneous structures that include high- and low-density biofilm regions, water-like voids, and solid-like inclusions. Channels larger than approximately 50 μm and connected to the bulk fluid were not visible. Both cluster and ring-like structures were observed with each granule source having a characteristic structural type. These structures, and their NMR relaxation behavior, were stable over several months of storage. These observations reveal the complex structures within aerobic granules from a range of sources and highlight the need for non-invasive characterization methods like NMR to be applied in the ongoing effort to correlate structure and function.

Aerobic granular sludge (AGS) technology allows simultaneous nitrogen, phosphorus, and carbon removal in compact wastewater treatment processes. To operate, design, and model AGS reactors, it is essential to properly understand the diffusive transport within the granules. In this study, diffusive mass transfer within full-scale and lab-scale AGS was characterized with nuclear magnetic resonance (NMR) methods. Self-diffusion coefficients of water inside the granules were determined with pulsed-field gradient NMR, while the granule structure was visualized with NMR imaging. A reaction-diffusion granule-scale model was set up to evaluate the impact of heterogeneous diffusion on granule performance. The self-diffusion coefficient of water in AGS was ∼70% of the self-diffusion coefficient of free water. There was no significant difference between self-diffusion in AGS from full-scale treatment plants and from lab-scale reactors. The results of the model showed that diffusional heterogeneity did not lead to a major change of flux into the granule (<1%). This study shows that differences between granular sludges and heterogeneity within granules have little impact on the kinetic properties of AGS. Thus, a relatively simple approach is sufficient to describe mass transport by diffusion into the granules.