Growth kinetics of aerobic granules developed in sequencing batch reactors

AIMS: This paper attempts to develop a kinetic model to describe the growth of aerobic granules developed under different operation conditions. METHODS AND RESULTS: A series of experiments were conducted by using four-column sequencing batch reactors to study the formation of aerobic granules under different conditions, e.g. organic loading rates, hydrodynamic shear forces and substrate N/COD ratios. A simple kinetic model based on the Linear Phenomenological Equation was successfully derived to describe the growth of aerobic granules. It was found that the growth of aerobic granules in terms of equilibrium size and size-dependent growth rate were inversely related to shear force imposed to microbial community, while a high organic loading favoured the growth of aerobic granules, leading to a large size granule. The effect of substrate N/COD ratio on the growth kinetics of aerobic granules was realized through change in microbial populations, and enriched nitrifying population in aerobic granules developed at high substrate N/COD ratio resulted in a low overall growth rate of aerobic granules. CONCLUSIONS: The proposed model can provide good prediction for the growth of aerobic granules indicated by the correlation coefficient >0.95. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model proposed could offer a useful tool for studying the growth kinetics of cell-to-cell immobilization process. The study confirmed that the growth of aerobic granules and biofilms are subject to a similar kinetic pattern. This work would also be helpful for better understanding the mechanism of aerobic granulation.     

Structure and stability of aerobic granules cultivated under different shear force in sequencing batch reactors

The cultivation of stable aerobic granules as well as granular structure and stability in sequencing batch reactors under different shear force were investigated in this study. Four column sequencing batch reactors (R1–R4) were operated under various shear force, in terms of superficial upflow air velocity of 0.8, 1.6, 2.4, and 3.2 cm s⁻¹, respectively. Aerobic granules were formed in all reactors in the experiment. It was found that the magnitude of shear force has an important impact on the granule stability. At shear force of 2.4 and 3.2 cm s⁻¹, granules can maintain a robust structure and have the potential of long-term operation. Granules developed in low shear force (R1, 0.8 cm s⁻¹ and R2, 1.6 cm s⁻¹) deteriorated to large-sized filamentous granules with irregular shape, loose structure and resulted in poor performance and operation instability. Granules cultivated under high shear force (R3, 2.4 cm s⁻¹ and R4, 3.2 cm s⁻¹) stabilized to clear outer morphology, dense and compact structure, and with good performance in 120 days operation. Fractal dimension (D_f) represents the internal structure of granules and can be used as an important indicator to describe the structure and stability of granules. Due to the combined effects of shear force and growth force, the mature granules developed in R3 and R4 also displayed certain differences in granular structure and characteristics.     

Influence of aeration intensity on mature aerobic granules in sequencing batch reactor

Aeration intensity is well known as an important factor in the formation of aerobic granules. In this research, two identical lab-scale sequencing batch reactors with aeration intensity of 0.8 (R1) and 0.2 m³/h (R2) were operated to investigate the characteristics and kinetics of matured aerobic granules. Results showed that both aeration intensity conditions induced granulation, but they showed different effects on the characteristics of aerobic granules. Compared with the low aeration intensity (R2), the aerobic granules under the higher aeration intensity (R1) had better physical characteristics and settling ability. However, the observed biomass yield (Y _obs) in R1 [0.673 kg mixed liquor volatile suspended solids (MLVSS)/kg chemical oxygen demand (COD)] was lower than R2 (0.749 kg MLVSS/kg COD). In addition, the maximum specific COD removal rates (q _max) and apparent half rate constant (K) of mature aerobic granular sludge under the two aeration intensities were at a similar level. Therefore, the matured aerobic granule system does not require to be operated in a higher aeration intensity, which will reduce the energy consumption.     

Characteristics and stability of aerobic granules cultivated with different starvation time

The characteristics of aerobic granules at steady state and the effects of starvation time on the stability of aerobic granules during the long-term operation were investigated in three sequencing batch reactors (SBRs R1–R3). The SBRs were operated with a cycle time of 1.5, 4.0, and 8.0 h, respectively, which resulted in a starvation time of 0.8, 3.3, and 7.3 h in three reactors, respectively. Results showed that aerobic granules were successfully cultivated in the three reactors, but the granules in R2 with a starvation time of 3.3 h showed the highest density and the best settleability at steady state. It is obvious that the starvation time has an optimum value in terms of settleability of granules. In addition, it was found that the coexistence of a minority of fluffy granules with smooth granules was the potential unstable factor in R1 with a starvation time of 0.8 h at the steady state. The sudden dominance of fluffy granules in R1 after the 160-day operation led to the operation failure of the reactor R1, whereas the granules in R2 with a starvation time of 3.3 h and R3 with a starvation time of 7.3 h showed good stability during the long-term operation. As short starvation time leads to the instability of granules, and long starvation time is not advisable for practical application due to low efficiency, starvation time should be controlled in a reasonable range.     

Quorum sensing unveils the sludge floccule-assisted stabilization of aerobic granules in granule-dominated sequencing batch reactors

Floccules are another major form of microbial aggregates in aerobic granular sludge systems. Previous studies mainly attributed the persistence of floccules to their relatively faster nutrient uptake and higher growth rate over aerobic granules; however, they failed to unravel the underlying mechanism of the long-term coexistence of these two aggregates. In this work, the existence and function of the floccules in an aerobic granule-dominated sequencing batch reactor were investigated from the view of quorum sensing (QS) and quorum quenching (QQ). The results showed that though the floccules were closely associated with the granules in terms of similar community structures (including the QS- and QQ-related ones), they exhibited a relatively higher QQ-related activity but a lower QS-related activity. A compatible proportion of floccules might be helpful to maintain the QS-related activity and keep the granules stable. In addition, the structure difference was demonstrated to diversify the QS- and QQ-related activities of the floccules and the aerobic granules. These findings could broaden our understanding of the interactions between the coexistent floccules and granules in aerobic granule-dominated systems and would be instructive for the development of the aerobic granular sludge process.     

Characterization and evaluation of aerobic granules in sequencing batch reactor

In order to investigate the aerobic granules cultured under alternating aerobic and anoxic conditions, a sequencing batch reactor (SBR) was operated without the presence of a carrier material. Nitrification and denitrification occurred alternately in the SBR operation, with an increased nitrification efficiency of up to 97% and a high chemical oxygen demand (COD) removal efficiency of up to 95%. It was observed that physical characteristics of granule play an important role in the performance of the SBR process. Light microscopy was used to observe the time dependent development of the granules in the SBR. Based on the microscopic observations, some floc-like sludges remained in the form of a mixture with granules for 30 days of operation. Even though various granule sizes had been formed in the reactor after 50 days, the granule sizes were primarily from 1 +/- 0.35 to 1.3 +/- 0.45 mm, rarely exceeding 2 mm. The granules were analyzed by a combination of microelectrodes and fluorescent in situ hybridization (FISH), which provides more detailed information on what happens inside the granules. Based on their results, ammonia oxidizing bacteria (AOB) existed primarily in the upper and middle layers of the granule. Assuming a first-order reaction for nitrification, most of the nitrification is likely to occur from the surface to 300 microm into the granular thickness.     

Development and characteristics of phosphorus-accumulating microbial granules in sequencing batch reactors

Phosphorus (P)-accumulating microbial granules were developed at different substrate P/chemical oxygen demand (COD) ratios in the range of 1/100 to 10/100 by weight in sequencing batch reactors. The soluble COD and PO4-P profiles showed that the granules had typical P-accumulating characteristics, with concomitant uptake of soluble organic carbon and the release of phosphate in the anaerobic stage, followed by rapid phosphate uptake in the aerobic stage. The size of P-accumulating granules exhibited a decreasing trend with the increase in substrate P/COD ratio, while the structure of the granules became more compact and denser as the substrate P/COD ratio increased. The P uptake by granules fell within the range of 1.9% to 9.3% by weight, which is comparable with uptake obtained in conventional enhanced biological phosphorus removal (EBPR) processes. It was further found that low aerobic respirometric activity of granules in terms of specific oxygen utilization rate favors P uptake by granules. The results presented would be useful for the further development of a novel granule-based EBPR technology.     

Causes and control of filamentous growth in aerobic granular sludge sequencing batch reactors

Poor long-term stability of aerobic granules developed in sequencing batch reactors (SBRs) remains a limitation to widespread use of aerobic granulation in treating wastewater. Filamentous growth has been commonly reported in aerobic granular sludge SBR. This review attempts to address the instability problem of aerobic granular sludge SBR from the perspective of filamentous growth in the system. The possible causes of filamentous growth are identified, including long retention times of solids, low substrate concentration in the liquid phase, high substrate gradient within the granule, dissolved oxygen deficiency in the granule, nutrient deficiency inside granule, temperature shift and flow patterns. Because of cyclic operation of aerobic granular sludge SBR and peculiarities of aerobic granules, various stresses can be present simultaneously and can result in progressive development of filamentous growth in aerobic granular sludge SBR. Overgrowth of filamentous bacteria under stress conditions appears to be a major cause of instability of aerobic granular sludge SBR. Specific recommendations are made for controlling filamentous growth.     

A unified theory for upscaling aerobic granular sludge sequencing batch reactors

Aerobic granular sludge sequencing batch reactors (SBR) are a promising technology for treating wastewater. Increasing evidence suggests that aerobic granulation in SBRs is driven by selection pressures exerted on microorganisms. Three major selection pressures have been identified as follows: settling time, volume exchange ratio and discharge time. This review demonstrates that these three major selection pressures can all be unified to one, the minimal settling velocity of bio-particles, that determines aerobic granulation in SBRs. The unified selection pressure theory is a useful guide for manipulating and optimizing the formation and characteristics of aerobic granules in SBRs. Furthermore, the unified theory provides a single engineering basis for scale up of aerobic granular sludge SBRs.     

Formation and long-term stability of nitrifying granules in a sequencing batch reactor

The formation and long-term stability of nitrifying granules in a sequencing batch reactor was investigated in this study. The results showed that nitrifying granules with a size of 240 microm and SVI of 40 ml g(-1) were formed on day 21 at a settling time of 10 min. Maintaining settling time at 15 min from day 57 to 183 did not affect the physical characteristics of sludge and the fraction of suspended floc in the sludge. In addition, nitrifying granules could tolerate the fluctuations of nitrogen loading rate from 0.72 to 1.8 g l(-1)d(-1) during 2 months without the change of physical characteristics. However, it was observed that complete nitrification to nitrate and partial nitrification to nitrite by sludge converted each other corresponding to the change of the influent NH4+-N concentration. Thus, an appropriate method is needed to maintain a stable complete nitrification or partial nitrification under the conditions with changing influent NH4+-N concentrations and nitrogen loading rates.     

The influence of short-term starvation on aerobic granules

Evidence shows that almost all aerobic granules can only be cultivated in sequencing batch reactor (SBR). Compared to continuous process, the unique feature of SBR is its cycle operation, which results in a periodical starvation in the reactor. So far, the effect of such a periodical starvation on aerobic granulation process remains unknown. Thus, this study investigated the responses of aerobic granules to the respective carbon-, nitrogen-, phosphorus-, potassium-starvation and also their collective effects in terms of cell surface hydrophobicity, surface zeta potential, extracelluar polysaccharides content, specific oxygen utilization rate and biomass growth. Results showed that short-term C-, N-, P- and K- starvations would pose negative effects on aerobic granules, e.g. reduce EPS content, inhibit microbial activity, weaken structural integrity and worsen settleability of aerobic granules. This study likely provides primary evidence that the substrate and nutrients starvation would not contribute to the stability of aerobic granules in a significant way.     

Effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors

Copper (Cu(II)) and nickel (Ni(II)) are often encountered in wastewaters. This study investigated the individual toxic effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors (SBRs). The biochemical properties of aerobic granules were characterized by extracellular polymeric substances (EPS) content, dehydrogenase activity, microbial community biodiversity, and SBR performance. One SBR was used as a control system, while another two received respective concentration of Cu(II) and Ni(II) equal to 5 mg/L initially and increased to 15 mg/L on day 27. Results showed that the addition of Cu(II) drastically reduced the biomass concentration, bioactivity, and biodiversity of aerobic granules, and certainly deteriorated the treatment performance. The toxic effect of Ni(II) on the biodiversity of aerobic granules was milder and the aerobic granular system elevated the level of Ni(II) toxicity tolerance. Even at a concentration of 15 mg/L, Ni(II) still stimulated the biomass yield and bioactivity of aerobic granules to some extent. The elevated tolerance seemed to be owed to the concentration gradient developed within granules, increased biomass concentration, and promoted EPS production in aerobic granular systems.     

Biodegradation and kinetics of aerobic granules under high organic loading rates in sequencing batch reactor

Biodegradation, kinetics, and microbial diversity of aerobic granules were investigated under a high range of organic loading rate 6.0 to 12.0 kg chemical oxygen demand (COD) m⁻³ day⁻¹ in a sequencing batch reactor. The selection and enriching of different bacterial species under different organic loading rates had an important effect on the characteristics and performance of the mature aerobic granules and caused the difference on granular biodegradation and kinetic behaviors. Good granular characteristics and performance were presented at steady state under various organic loading rates. Larger and denser aerobic granules were developed and stabilized at relatively higher organic loading rates with decreased bioactivity in terms of specific oxygen utilization rate and specific growth rate (μ _overall) or solid retention time. The decrease of bioactivity was helpful to maintain granule stability under high organic loading rates and improve reactor operation. The corresponding biokinetic coefficients of endogenous decay rate (k _d), observed yield (Y _obs), and theoretical yield (Y) were measured and calculated in this study. As the increase of organic loading rate, a decreased net sludge production (Y _obs) is associated with an increased solid retention time, while k _d and Y changed insignificantly and can be regarded as constants under different organic loading rates.     

Microbial community of aerobic granules for ammonium and sulphide removal in a sequencing batch reactor

Aerobic granules for sulphide and ammonium removal were cultivated in a sequencing batch reactor, and the microbial community of the aerobic granules was investigated by denaturing gradient gel electrophoresis. The loading rate increased from 0.15 to 0.9 kg S2? m?3 d?1, and the removal efficiencies of sulphide, chemical oxygen demand, and NH4 +-N were higher than 99, 80, and 98%, respectively. However, sludge settleability became poorer when the loading rate exceeded 0.3 kg S2? m?3 d?1. The denitrifying bacteria in the aerobic granules were Thauera sp., Pseudomonas alcaligenes, and uncultured planctomycetes, indicating that multiple N-removing processes occurred simultaneously in the aerobic granules. These processes could include nitrification and denitrification, aerobic denitrification, and anaerobic ammonia oxidation. Sludge settleability became poorer because of the overgrowth of uncultured Thiothrix sp.     

Simulation of wastewater treatment by aerobic granules in a sequencing batch reactor based on cellular automata

In the present paper, aerobic granules were developed in a sequencing batch reactor (SBR) using synthetic wastewater, and 81 % of granular rate was obtained after 15-day cultivation. Aerobic granules have a 96 % BOD removal to the wastewater, and the reactor harbors a mount of biomass including bacteria, fungi and protozoa. In view of the complexity of kinetic behaviors of sludge and biological mechanisms of the granular SBR, a cellular automata model was established to simulate the process of wastewater treatment. The results indicate that the model not only visualized the complex adsorption and degradation process of aerobic granules, but also well described the BOD removal of wastewater and microbial growth in the reactor. Thus, CA model is suitable for simulation of synthetic wastewater treatment. This is the first report about dynamical and visual simulation of treatment process of synthetic wastewater in a granular SBR.     

Bacterial diversity and function of aerobic granules engineered in a sequencing batch reactor for phenol degradation

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of beta-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.     

The role of cellular polysaccharides in the formation and stability of aerobic granules

AIMS: This paper attempts to investigate the role of cellular polysaccharides in the formation and stability of aerobic granules. METHODS AND RESULTS: Three column sequential aerobic sludge blanket reactors (R1, R2 and R3) were operated at a superficial air upflow velocity of 0.3 cm s(-1), 1.2 cm s(-1) and 2.4 cm s(-1), respectively. Aerobic granules appeared at cycle 42 in R2 and R3 with a mean size of 0.37 mm in R2 and 0.35 mm in R3, however, aerobic granulation was not observed in R1. After the formation of aerobic granules, the sludge volume index (SVI) decreased to 55 ml g(-1) in R2 and 46 ml g(-1) in R3. Aerobic granulation was concurrent with a sharp increase of cellular polysaccharides normalized to cellular proteins, which increased from 5.7 to 13.0 mg per mg proteins in R2, and 7.5-13.9 mg per mg protein in R3. The content of polysaccharides in aerobic granules was 2-3 times higher than that in the bioflocci cultivated in R1. The disappearance of aerobic granules in R2 was tightly coupled to a drop in cellular polysaccharides. After the reappearance of bioflocci in R2, the content of cellular polysaccharides were found to be restored to the level observed in R1. CONCLUSION: It appears that the production of cellular polysaccharides could be stimulated by hydrodynamic shear force and contributes to the formation and stability of aerobic granules. SIGNIFICANCE AND IMPACT OF THE STUDY: It is expected that this study would provide useful information for better understanding the mechanisms of aerobic granulation.     

Essential roles of eDNA and AI-2 in aerobic granulation in sequencing batch reactors operated at different settling times

Settling time has been considered as one of the most effective selection pressures for aerobic granulation in sequencing batch reactors (SBRs), i.e., poorly settleable bioparticles would be washed out from SBRs, and the heavy and good settling ones would be retained at a shorter setting time. However, its biological implication remains unclear. This study investigated the microbiological mechanisms of aerobic granulation at different settling times. It provided experimental evidence for the first time showing that a shorter settling time could enhance release of extracellular DNA through cell lysis, which in turn initiated microbial aggregation leading to increased biomass size and density, while AI-2-mediated quorum sensing was found not to be involved in initial aggregation. It was further shown that the AI-2-mediated quorum sensing system was activated to regulate the growth and maturation of aerobic granules when the biomass density reached a threshold of 1.025 g ml⁻¹. It appears from this study that a short settling time of SBR would induce microbiological and physiological responses of bacteria which are required at different stages of aerobic granulation and provide new insights into biological mechanisms of settling time-triggered aerobic granulation.     

Hydraulic selection pressure-induced nitrifying granulation in sequencing batch reactors

The effect of hydraulic selection pressure on the development of nitrifying granules was investigated in four column-type sequencing batch reactors (SBR). The nature of SBR is cycle operation, thus SBR cycle time can serve as a main hydraulic selection pressure imposed on the microbial community in the system. No nitrifying granulation was observed in the SBR operated at the longest cycle time of 24 h, due to a very weak hydraulic selection pressure, while the washout of nitrifying sludge was found in the SBR run at the shortest cycle time of 3 h, and led to a failure of nitrifying granulation. Excellent nitrifying granules with a mean diameter of 0.25 mm and specific gravity of 1.014 were developed in a SBR operated at cycle times of 6 h and 12 h, respectively. The results further showed that a short cycle time would stimulate microbial activity, production of cell polysaccharides and also improve the cell hydrophobicity. These hydraulic selection pressure-induced microbial changes favour the formation of nitrifying granules. This work, probably for the first time, shows that nitrifying granules can be developed at a proper hydraulic selection pressure in terms of SBR cycle time. Nitrifying granulation is a novel biotechnology which has a great potential for wastewater nitrification.