Domestic wastewater is treated prior to its return to natural water bodies, to minimize its polluting effect. Biological wastewater treatment removes organic matter and nutrients from the wastewater, by employing the activity of microorganisms, which consume polluting compounds present in wastewater to grow. One of such technologies is aerobic granular sludge (AGS), which consists of self-immobilized microorganisms growing in spherical biofilms. The granular structure facilitates the separation between treated water and the biomass due to its excellent settling properties. This way, energy and space are saved in comparison to flocculent sludge-based treatment. Despite its many advantages, the granular structure can pose some challenges too, particularly regarding the degradation of polymeric substrates. The higher mass-transfer resistance in granules compared to flocs challenges the degradation of these substrates, which have a size spanning from a few kDa to several micrometres. Polymeric substrates, furthermore, need to undergo hydrolysis before microorganisms can take them up, which is generally a slow process. Most AGS applications rely on microbial selection driven by the application of a sequencing batch reactor (SBR) cycle. The cycle consists of an anaerobic substrate feeding and a subsequent aerobic starvation period, which selects for intracellular polymer-storing organisms, such as polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO). Substrates that experience high mass-transfer limitation and low degradation rates may interfere with the microbial selection strategy applied to AGS, especially when they are not (fully) taken up in the anaerobic feeding period and continue degrading aerobically in the next cycle phase. Some lab-scale studies have reported detrimental effects of polymeric substrates in AGS structure and activity, while others have managed to maintain a stable granule bed and suggest that the microbial utilization of polymeric substrates can contribute to good nutrient removal. The degradation of polymeric substrates by full-scale aerobic granules is still poorly understood. @en

Aerobic granular sludge (AGS) is an advanced biofilm-based technology for wastewater treatment. Diffusion of substrates into the granules is a key aspect of this technology. Domestic wastewater contains soluble organic substrates of different sizes that could potentially diffuse into the granules. In this study, the relation between the molecular weight of a substrate and its diffusion coefficient within the granule was studied with model substrates (polyethylene glycols (PEGs) with a molecular weight between 62 and 10 000 Da). The diffusion coefficients of the model substrates within granules from a full-scale installation were measured with the ‘transient uptake of a non-reactive solute’ method. The diffusion coefficients in the granules were not significantly different from the diffusion coefficients in water, at least up to 4000 Da molecular weight. This indicates that these PEGs were not obstructed by the granule matrix. The 10 kDa PEG behaved differently from the lighter PEGs, as it could not penetrate the entire granule. Furthermore, the granule structure was characterized with Environmental Scanning Electron Microscopy (ESEM). The granules displayed an open structure with large macropores and semi-solid regions, which contained microbial cells. The diffusion results suggest that most diffusing molecules were unobstructed in the macropores and barely obstructed in the semi-solid regions. Only the diffusion of the 10 kDa PEG seemed to be hindered by the semi-solid regions, but not by the macropores. Lastly, the apparent molecular weight distribution of domestic wastewater soluble COD was determined with ultrafiltration membranes of 100, 10, and 1 kDa molecular weight cut-off. The influent fractionation revealed that a large part (61-69%) of the influent soluble COD was lighter than 1 kDa. As molecules lighter than 1 kDa diffuse easily, the majority of the influent soluble COD can be considered as diffusible COD. These findings provide new insight into the availability of influent COD for granular sludge.@en

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.

@en

High concentrations of particulate COD (pCOD) in the influent of aerobic granular sludge (AGS) systems are often associated to small granule diameter and a large fraction of flocculent sludge. At high particulate concentrations even granule stability and process performance might be compromised. However, pilot-or full-scale studies focusing on the effect of real wastewater partic-ulates on AGS are scarce. This study describes a 3-month period of increased particulate loading at a municipal AGS wastewater treatment plant. The pCOD concentration of the influent increased from 0.5 g COD/L to 1.3 g COD/L, by adding an untreated slaughterhouse wastewater source to the influent. Sludge concentration, waste sludge production and COD and nutrient removal performance were monitored. Furthermore, to investigate how the sludge acclimatises to a higher influent particulate content, lipase and protease hydrolytic activities were studied, as well as the microbial community composition of the sludge. The composition of the granule bed and nutrient removal efficiency did not change considerably by the increased pCOD. Interestingly, the biomass-specific hydrolytic activities of the sludge did not increase during the test period either. However, already during normal operation the aerobic granules and flocs exhibited a hydrolytic potential that ex-ceeded the influent concentrations of proteins and lipids. Microbial community analysis also revealed a high proportion of putative hydrolysing and fermenting organisms in the sludge, both during normal operation and during the test period. The results of this study highlight the robust-ness of the full-scale AGS process, which can bear a substantial increase in the influent pCOD concentration during an extended period.

@en

Complex substrates, like proteins, carbohydrates, and lipids, are major components of domestic wastewater, and yet their degradation in biofilm-based wastewater treatment technologies, such as aerobic granular sludge (AGS), is not well understood. Hydrolysis is considered the rate-limiting step in the bioconversion of complex substrates, and as such, it will impact the utilization of a large wastewater COD (chemical oxygen demand) fraction by the biofilms or granules. To study the hydrolysis of complex substrates within these types of biomass, this paper investigates the anaerobic activity of major hydrolytic enzymes in the different sludge fractions of a full-scale AGS reactor. Chromogenic substrates were used under fully mixed anaerobic conditions to determine lipase, protease, α-glucosidase, and β-glucosidase activities in large granules (>1 mm in diameter), small granules (0.2–1 mm), flocculent sludge (0.045–0.2 mm), and bulk liquid. Furthermore, composition and hydrolytic activity of influent wastewater samples were determined. Our results showed an overcapacity of the sludge to hydrolyze wastewater soluble and colloidal polymeric substrates. The highest specific hydrolytic activity was associated with the flocculent sludge fraction (1.5–7.5 times that of large and smaller granules), in agreement with its large available surface area. However, the biomass in the full-scale reactor consisted of 84% large granules, making the large granules account for 55–68% of the total hydrolytic activity potential in the reactor. These observations shine a new light on the contribution of large granules to the conversion of polymeric COD and suggest that large granules can hydrolyze a significant amount of this influent fraction. The anaerobic removal of polymeric soluble and colloidal substrates could clarify the stable granule formation that is observed in full-scale installations, even when those are fed with complex wastewaters. Key points: • Large and small granules contain >70% of the hydrolysis potential in an AGS reactor. • Flocculent sludge has high hydrolytic activity but constitutes <10% VS in AGS. • AGS has an overcapacity to hydrolyze complex substrates in domestic wastewater. <!-- Query ID="Q2" Text=" Graphical abstract contains text below the minimum required font size of 6pts inside the artwork, and there is no sufficient space available for the text to be enlarged. Please provide replacement figure file." -->Graphical abstract: [Figure not available: see fulltext.]@en