How to cultivate aerobic granular sludge from wastewater

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Aerobic granular sludge is a new technology in the biological treatment of wastewater. Compared to the flocs of activated sludge used in the currently prevalent activated sludge process, aerobic granular sludge has several advantages: under certain conditions, activated sludge flocs can transform into granules, which settle much faster in water than ordinary flocs. Therefore, when using aerobic granular sludge for wastewater treatment, the biomass concentration in the aeration tank can be significantly increased, while the sedimentation time can be greatly reduced. In conventional activated sludge processes, the concentration of activated sludge in the aeration tank is approximately 3,000 mg·L⁻¹, with a sedimentation time ranging from 30 minutes to 2 hours. However, with aerobic granular sludge technology, this concentration can reach 10,000–14,000 mg·L⁻¹, and the sedimentation time is merely 1–3 minutes. Compared to anaerobic granular sludge, which is commonly employed for treating wastewater with high concentrations of pollutants or hard-to-degrade substances, the cultivation period for aerobic granular sludge is only about one week to one month—far shorter than the six-month startup period required for anaerobic granular sludge. Thus, aerobic granular sludge technology holds great promise for bringing about breakthroughs in current wastewater treatment methods.

However, research on the granulation of aerobic sludge is still in its infancy; there remains a lack of in-depth studies regarding the formation process and mechanisms of aerobic granular sludge, the effects of various environmental factors on it, as well as its microbiology. Moreover, most existing research on aerobic granular sludge has been conducted on a laboratory scale, utilizing synthetic wastewater containing relatively high concentrations of organic matter (such as glucose) as a substrate. There are few studies focusing on cultivating aerobic granular sludge using municipal wastewater with low organic content. Municipal wastewater contains various types of pollutants, yet its COD levels are generally low—typically below 200 mg·L⁻¹. Currently, traditional activated sludge processes are widely used for treating such wastewater; although they achieve decent treatment results, these systems often require large areas of land, involve high construction costs, generate substantial amounts of excess sludge, incur high operating expenses, and are prone to sludge bulking.

In this study, a pilot-scale experimental setup was developed to cultivate aerobic granular sludge using actual municipal wastewater. Modern analytical techniques such as confocal laser scanning microscopy and X-ray diffraction were employed to analyze the characteristics of the resulting granules, with the aim of laying a foundation for the practical application of aerobic granular sludge technology.

1 Materials and Methods
1.1 Experimental Setup
The pilot experiment utilized a cylindrical SBR reactor. Its upper portion was made of acrylic glass, while the lower part was constructed from steel and coated with anti-corrosion paint. The reactor had an inner diameter of 1 m, an effective height of 4.5 m, and an effective volume of 3.5 m³. Compressed air was supplied via an air compressor, and the airflow rate was regulated by a flowmeter; the airflow rate was set at 12.5 m³·h⁻¹, resulting in a gas velocity of 0.44 cm·s⁻¹ along the inner surface of the reactor. This gas velocity was calculated using the following formula:
Gas velocity (cm·s⁻¹) = Gas flow rate (m³·s⁻¹) / Cross-sectional area of reactor (m²) × 100

The experimental setup is illustrated in Figure 1. Each cycle within the SBR lasted 180 minutes: 8 minutes for influent introduction, 160 minutes for aeration, 6 minutes for sedimentation, and 6 minutes for effluent discharge. The entire operational sequence was controlled via a microcomputer-based timer. Synthetic wastewater was pumped into the reactor from its upper section via a metering pump, while effluent was discharged through an outlet located midway up the reactor; the effluent ratio was 50%. Throughout each 3-hour cycle, the dissolved oxygen (DO) concentration within the reactor remained above 2 mg·L⁻¹.
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Figure 1 Schematic diagram of the pilot-scale setup: 1. Raw water tank; 2. Influent pump; 3. Microcomputer-based timer; 4. Air compressor; 5. Airflow meter; 6. Diffuser; 7. Effluent pump; 8. Outlet

1.2 Inoculation and Cultivation
The inoculum used was return sludge obtained from the secondary sedimentation tank of an actual wastewater treatment plant. This sludge exhibited a loose, yellow-colored floccular structure; its properties are detailed in Table 1. After being inoculated, the sludge was aerated for 24 hours, followed by 30 minutes of sedimentation; the supernatant was then removed so that the final volume of inoculum accounted for 50% of the total volume. Subsequently, wastewater was added until the reactor reached its normal water level. Following inoculation, the initial sludge concentration within the reactor was approximately 3,000 mg·L⁻¹.

The test wastewater utilized was actual municipal wastewater sourced from a treatment plant; its characteristics are listed in Table 2.
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Table 1 Properties of the inoculum sludge
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Table 2 Characteristics of the influent wastewater

1.3 Analytical Methods
(1) Conventional Analyses
COD and NH₄⁺-N levels were determined via rapid closed-vial spectrophotometry. NO₂⁻-N, NO₃⁻-N, TP, mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS), and suspended solids (SS) in effluent were measured using standard methodologies. Images were captured using an Olympus CX31 optical microscope paired with a digital camera produced by Olympus.

(2) CLSM Analysis
Freeze-sectioning: Aerobic granular sludge samples were extracted from the reactor, washed with PBS, and placed in Tissue-Tek OCT freezing medium (Miles, Elkhart, IN); they were then frozen at -40°C overnight. These frozen samples were sliced into sections measuring 50 μm thickness using a cryostat (CM 1510-Cryostat, Leica, Germany).

CLSM analysis: Bacterial cells within these sections were stained using the nucleic acid dye SYTO9 (25 mmol·L⁻¹, Molecular Probes, Eugene, OR), while extracellular polymeric substances (EPS) were stained using ConA-TRITC fluorescent dye (250 mg·L⁻¹, Sigma). After 20 minutes of staining, the samples were rinsed with PBS. Finally, these stained samples were examined under a confocal laser scanning microscope (CLSM, LSM 5 Pascal, Zeiss, Jena, Germany).

(3) Analysis of Inorganic Components in Aerobic Granular Sludge
An X-ray diffraction (XRD) analyzer was employed to determine the inorganic composition of aerobic granular sludge. The procedure involved heating the samples at 550°C for over 30 minutes, cooling and drying them, then grinding them into powder form. An X-ray powder diffractometer (Bruker D8 Advance; Cu-Kα radiation, LynxEye detector; tube voltage: 40 kV; current: 40 mA; angular range: 10°–80°, step size: 0.02°; scan speed: 0.3 s/step) was used to collect diffraction patterns. Data analysis was performed using Eva XRD Pattern Processing software (Bruker Co. Ltd.).

2 Results and Analysis
2.1 Formation of Aerobic Granular Sludge
Throughout the granulation process, changes in the morphology of the sludge within the reactor were observed using an optical microscope; these changes are depicted in Figure 2. It is evident that as cultivation progressed, initially dispersed floccular sludge gradually transformed into small, irregularly shaped granules; these subsequently grew into larger, more uniformly shaped granules. Eventually, well-defined, dark brown-colored, ellipsoidal aerobic granular sludge formed. The proportion of granular sludge within the reactor steadily rose, increasing from roughly 20% after 40 days to around 85% after 100 days.
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Figure 2 Morphological changes of aerobic granular sludge

During cultivation, the sedimentation time within the reactor decreased progressively from 30 minutes to just 6 minutes. As this time was reduced, excessive sludge removal occurred, causing the MLSS concentration to drop from 2.13 g·L⁻¹ to 0.94 g·L⁻¹ (Figure 3). Consequently, the granules gained access to greater quantities of nutrients, prompting a gradual rise in MLSS levels as granulation advanced. By day 40, the average particle size had increased noticeably, indicating near-complete maturation; at this point, individual granules could reach sizes of approximately 1.0 mm. Meanwhile, the MLSS concentration within the reactor stood at roughly 1.2 g·L⁻¹.

The influent to the wastewater treatment plant originated from a river. On day 75, heavy rainfall led to a sharp surge in suspended solids content within the riverwater, thereby elevating the MLSS concentration in the reactor. Once the precipitation ceased, this concentration returned to its baseline level.
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Figure 3 Variations in MLSS and MLVSS concentrations over time within the pilot-scale SBR reactor

2.2 Removal of Pollutants
Figure 4 illustrates how SS concentrations in effluent varied over time during the experiment. As cultivation progressed, the proportion of granular sludge within the reactor continued to increase, leading to a corresponding decline in SS levels in effluent. Nevertheless, compared to Class A standards stipulated for effluent discharged from wastewater treatment plants (where permissible SS