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.     

Mathematical modeling of aerobic granular sludge: A review

Aerobic granulation may play an important role in the field of wastewater treatment due to the advantages of aerobic granules compared to the conventional sludge flocs, such as denser structure, better settleability and ensured solid-effluent separation, higher biomass concentration, and greater ability to withstand shock loadings, which is promising for a full-scale implementation. As an aid for this implementation, mathematical modeling would be an invaluable tool. In this paper, the existing mathematical models available in literature concerning aerobic granule systems are reviewed, including the modeling of the dynamic facets of the aerobic granulation process, the mass transfer and detachment in aerobic granules, the granule-based sequencing batch reactor, the fate of microbial products in granules, and the multi-scale modeling of aerobic granular sludge. An overview of the parameters used in the aerobic granular modeling approaches is also presented. Our growing knowledge on mathematical modeling of aerobic granule might facilitate the engineering and optimization of aerobic granular sludge technology as one of the most promising techniques in the biological wastewater treatment.     

Aerobic granular sludge: recent advances

Aerobic granulation, a novel environmental biotechnological process, was increasingly drawing interest of researchers engaging in work in the area of biological wastewater treatment. Developed about one decade ago, it was exciting research work that explored beyond the limits of aerobic wastewater treatment such as treatment of high strength organic wastewaters, bioremediation of toxic aromatic pollutants including phenol, toluene, pyridine and textile dyes, removal of nitrogen, phosphate, sulphate and nuclear waste and adsorption of heavy metals. Despite this intensive research the mechanisms responsible for aerobic granulation and the strategy to expedite the formation of granular sludge, and effects of different operational and environmental factors have not yet been clearly described. This paper provides an up-to-date review on recent research development in aerobic biogranulation technology and applications in treating toxic industrial and municipal wastewaters. Factors affecting granulation, granule characterization, granulation hypotheses, effects of different operational parameters on aerobic granulation, response of aerobic granules to different environmental conditions, their applications in bioremediations, and possible future trends were delineated. The review attempts to shed light on the fundamental understanding in aerobic granulation by newly employed confocal laser scanning microscopic techniques and microscopic observations of granules.     

Advances in aerobic granule formation and granule stability in the course of storage and reactor operation

Aerobic granulation is drawing increasing global interest in a quest for an efficient and innovative technology in wastewater treatment. Developed less than two decades ago, extensive research work on aerobic granulation has been reported. The instability of the granule, which is one of the main problems that hinder practical application of aerobic granulation technology, is still to be resolved. This paper presents a review of the literature in aerobic granulation focusing on factors that influence granule formation, granule development and their stability in the context of sludge granulation. The review attempts to shed light on the potential of developing granules with adequate structural stability for practical applications. The possibilities and perspective of using stored granule as inoculums for rapid startup, and as microbial supplement to enhance treatment of bioreactor systems are also discussed.     

State of the art of aerobic granulation in continuous flow bioreactors

In the wake of the success of aerobic granulation in sequential batch reactors (SBRs) for treating wastewater, attention is beginning to turn to continuous flow applications. This is a necessary step given the advantages of continuous flow treatment processes and the fact that the majority of full-scale wastewater treatment plants across the world are operated with aeration tanks and clarifiers in a continuous flow mode. As in SBRs, applying a selection pressure, based on differences in either settling velocity or the size of the biomass, is essential for successful granulation in continuous flow reactors (CFRs). CFRs employed for aerobic granulation come in multiple configurations, each with their own means of achieving such a selection pressure. Other factors, such as bioaugmentation and hydraulic shear force, also contribute to aerobic granulation to some extent. Besides the formation of aerobic granules, long-term stability of aerobic granules is also a critical issue to be addressed. Inorganic precipitation, special inocula, and various operational optimization strategies have been used to improve granule long-term structural integrity. Accumulated studies reviewed in this work demonstrate that aerobic granulation in CFRs is capable of removing a wide spectrum of contaminants and achieving properties generally comparable to those in SBRs. Despite the notable research progress made toward successful aerobic granulation in lab-scale CFRs, to the best of our knowledge, there are only three full-scale tests of the technique, two being seeded with anammox-supported aerobic granules and the other with conventional aerobic granules; two other process alternatives are currently in development. Application of settling- or size-based selection pressures and feast/famine conditions are especially difficult to implement to these and similar mainstream systems. Future research efforts needs to be focused on the optimization of the granule-to-floc ratio, enhancement of granule activity, improvement of long-term granule stability, and a better understanding of aerobic granulation mechanisms in CFRs, especially in full-scale applications.     

Specific aerobic granules can be developed in a completely mixed tank reactor by bioaugmentation using micro-mycelial pellets of Phanerochaete chrysosporium

Aerobic granules were firstly developed in a completely mixed tank reactor (CMTR) by seeding micro-mycelial pellets (MMPs) of Phanerochaete chrysosporium. During phenol wastewater treatment, sludge granulation rate reached 67 % after 15-day operation. The granules in CMTR are different from aerobic granules described in literature in morphology, and a majority of them are rod-shaped or rodlike sludge besides spherical granules. The polymorphic granules, having no essential difference with aerobic granules previously reported, achieve advantages over conventional activated sludge in settling ability, biomass concentration, density, integrity coefficient and removal ability to phenol wastewater. The optimized parameters for sludge granulation in CMTR including temperature, inoculum quantity, rotary speed and superficial air upflow velocity are 30 °C, 5–7 g/l, 150 rpm, and 0.5 cm/s, respectively. Analysis on sludge granulation mechanism indicates that MMPs not only result in the formation of aerobic granules containing MMPs as nuclei, but also induce the formation of biogranules which do not have MMP at their cores. The work challenges the general belief that the homogenous circular flow pattern of microbial aggregates is necessary for aerobic sludge granulation.     

Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.     

Effects of Cycle Time on Properties of Aerobic Granules in Sequencing Batch Airlift Reactors

Summary The granulation and properties of aerobic sludge were studied in two sequencing batch airlift reactors (SBARs). The synthetic wastewater in the two reactors had initially different levels of COD (400 mg l⁻¹ in R1 and 1600 mg l⁻¹ in R2). A hydraulic cycle time of 3 and 12 h was conducted in the reactors R1 and R2, respectively and the process of granulation was observed by optical microscopy. It was found that the course of granulation at a cycle time of 3 h in R1 was shorter than that at cycle time of 12 h in R2 and the properties of aerobic granules were distinct in the reactors due to the different hydraulic cycle time. Under a cycle time of 3 h, granule diameter was around 1.0–2.0 mm, VSS ratio was 92.08% with stronger granule strength; under a cycle time of 12 h, granule diameter was around 0.5–1.0 mm, VSS ratio was 83.92% with weaker granule strength. In addition, the morphology of microorganisms in granules was obviously dissimilar when the hydraulic cycle time was different. It was concluded that the hydraulic cycle time plays a crucial role in the granulation and properties of aerobic granules. It is expected that the experimental findings will provide useful information on factors affecting aerobic granulation.     

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.     

State of the art of biogranulation technology for wastewater treatment

Biogranulation technology developed for wastewater treatment includes anaerobic and aerobic granulation processes. Anaerobic granulation is relatively well known, but research on aerobic granulation commenced only recently. Many full-scale anaerobic granular sludge units have been operated worldwide, but no report exists of similar units for aerobic granulation. This paper reviews the fundamentals and applications of biogranulation technology in wastewater treatment. Aspects discussed include the models of biogranulation, major factors influencing biogranulation, characteristics of biogranules, and their industrial applications. This review hopes to provide a platform for developing novel granules-based bioreactors and devising a unified interpretation of the formation of anaerobic and aerobic granules under various operation conditions.     

Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor

AIMS: This paper attempts to provide visual evidence of how aerobic granulation evolves in sequential aerobic sludge blanket reactors. METHODS AND RESULTS: A series of experiments were conducted in two column-type sequential aerobic sludge reactors fed with glucose and acetate as sole carbon source, respectively. The evolution of aerobic granulation was monitored using image analysis and optical and scanning electron microscopy. The results indicated that the formation of aerobic granules was a gradual process from seed sludge to compact aggregates, further to granular sludge and finally to mature granules with the sequential operation proceeding. Glucose- and acetate-fed granules have comparable characteristics in terms of settling velocity, size, shape, biomass density and microbial activity. However, the microbial diversity of the granules was associated with the carbon source supplied. In this work, an important aerobic starvation phase was identified during sequential operation cycles. It was found that periodical aerobic starvation was an effective trigger for microbial aggregation in the reactor and further strengthened cell-cell interaction to form dense aggregates, which was an essential step of granulation. The periodical starvation-induced aggregates would finally be shaped to granules by hydrodynamic shear and flow. CONCLUSION: Aerobic granules can be formed within 3 weeks in the systems. The periodical starvation and hydrodynamic conditions would play a crucial role in the granulation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic granules have excellent physical characteristics as compared with conventional activated sludge flocs. This research could be helpful for the development of an aerobic granule-based novel type of reactor for handling high strength organic wastewater.     

The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation

Quorum sensing (QS) through signal chemical molecules is known to be essential to bacterial adhesion and biofilm formation. In this study, the QS ability of aerobic granules—a special form of biofilms used for biological wastewater treatment—was investigated and compared with that of conventional activated sludge flocs. A novel sectional membrane bioreactor was used together with a flow-cell to evaluate the possible influence of signal chemicals produced by the source sludge on the growth mode of bacterial cells. The results demonstrate the apparent production of QS chemicals from granules and its impact on initial cell attachment and granule formation. When granules were used as the signal-producing biomass, the attached-growth mode was dominant for the free cells, and the biofilm formation rate in the flow-cell was about ten times faster than in cases which used activated sludge as the signal source biomass. In addition, the intracellular extract from mature granules significantly accelerated the sludge granulation process. It is argued that the production and expression of QS signal chemicals from granules and granule precursors might have induced the gene expression of bacteria in suspension for attached growth rather than suspended growth, leading to granule formation and its stable structure.     

Effect of iron ions (Fe<Superscript>2+</Superscript>, Fe<Superscript>3+</Superscript>) on the formation and structure of aerobic granular sludge

Aerobic granulation is a promising technology for wastewater treatment, but problems regarding its formation and stability need to be solved. Divalent metal ions, especially Ca²⁺, Mg²⁺ and Mn²⁺, have been demonstrated to play an important role in the process of aerobic granulation. Here, we studied whether iron ions can affect aerobic granulation. Granular sludge formed without iron ion addition (<0.02 mg Fe²⁺ L^?1) was fluffy and had a finger-type structure and filamentous out-growth. The addition of iron ions to concentrations of 1 and 10 mg Fe²⁺ L^?1 repressed the finger-type structure and filamentous out-growth. The results show that chemical precipitation in the granules with iron ion addition was higher than that in the granules without ferrous addition. The amount of precipitates was higher inside the granules than outside. This study demonstrates that iron ions (Fe²⁺/Fe³⁺) increase the size and stability of aerobic granular sludge but do not affect the granulation time, which is the time that the first granular sludge is observed. The study shows that aerobic granular sludge technology can be confidently applied to actual wastewater containing a high concentration of iron compounds.     

Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria

This study investigated the feasibility of improving the stability of aerobic granules through selecting slow-growing nitrifying bacteria. For this purpose, four sequencing batch reactors were operated at different substrate N/COD ratios ranging from 5/100 to 30/100. Results showed that aerobic granules formed in all four reactors, and aerobic granulation was a gradual process evolving from the dispersed seed sludge to mature and stable granules, and the whole granulation process could be divided into three phases, i.e. acclimation phase, granulation followed by granule maturation. The observed growth rate and mean size of mature aerobic granules were found to decrease as the substrate N/COD ratio was increased, while nitrifying population was enriched markedly in aerobic granules developed at high substrate N/COD ratios. The enriched nitrifying population in aerobic granules was responsible for the observed low growth rate of aerobic granules. It seems certain that the substrate N/COD ratio is an important factor in selecting nitrifying bacteria in aerobic granules. Aerobic granules with low growth rates showed strong structure and good settleability in terms of specific gravity, SVI and cell hydrophobicity that further lead to high stability as compared to those having high growth rates. This study demonstrated that the selection of slow-growing nitrifying bacteria through controlling substrate N/COD ratio would be a useful strategy for improving the stability of aerobic granules.     

Bacterial community dynamics in long‐term operation of a pilot plant using aerobic granular sludge to treat pig slurry

Aerobic granular sludge represents an interesting approach for simultaneous organic matter and nitrogen removal in wastewater treatment plants. However, the information about microbial communities in aerobic granular systems dealing with industrial wastewater like pig slurry is limited. Herein, bacterial diversity and dynamics were assessed in a pilot scale plant using aerobic granular sludge for organic matter and nitrogen elimination from swine slurry during more than 300 days. Results indicated that bacterial composition evolved throughout the operational period from flocculent activated sludge, used as inoculum, to mature aerobic granules. Bacterial diversity increased at the beginning of the granulation process and then declined due to the application of transient organic matter and nitrogen loads. The operational conditions of the pilot plant and the degree of granulation determined the microbial community of the aerobic granules. Brachymonas, Zoogloea and Thauera were attributed with structural function as they are able to produce extracellular polymeric substances to maintain the granular structure. Nitrogen removal was justified by partial nitrification (Nitrosomonas) and denitrification (Thauera and Zoogloea), while Comamonas was identified as the main organic matter oxidizing bacteria. Overall, clear links between bacterial dynamics and composition with process performance were found and will help to predict their biological functions in wastewater ecosystems improving the future control of the process. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1212–1221, 2016     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Characterization of aerobic granules by microbial density at different COD loading rates

Aerobic granules were cultivated under temporal alternating aerobic and anoxic conditions without the presence of a carrier material in a sequencing batch reactor (SBR) with a high column height/column diameter ratio. The reactor was operated for 6h per cycle (aerobic: 4.75 h, anoxic: 1.25 h). To determine a new parameter for the definition of aerobic granules, a protocol of 4,6-diamidino-2-phenylindole hydrochloride staining and fluorescence image processing was developed. The d(tm) analysis showed that the increase in the chemical oxygen demand (COD) loading rate promoted no more growth of the aerobic granules. It was inconsistent with the results of the analysis of the sludge volume index (SVI) value but matched well with the results of the COD and nitrogen removal of the SBR and the particle size distribution by LS-PSA. The optimum COD loading rate for aerobic granulation in the SBR was 2.52 kg/m(3)d. When d(tm) was correlated with the biomass concentration and the SVI value during the period of granule formation, d(tm) could be used as a more sensitive and accurate parameter for classifying aerobic granules and optimizing the operational conditions for aerobic granulation processes.     

Microbial population dynamics during sludge granulation in an anaerobic-aerobic biological phosphorus removal system

The evolution of a microbial community was investigated during sludge granulation using a wide range of micro-scale and molecular biology techniques. Experimental results demonstrate that polyphosphate-accumulating granules were successfully cultured during the anaerobic/aerobic cycle. Improvement in sludge sedimentation performance occurred prior to the formation of granular sludge and was not affected by change in granule size. Rod-shaped and filamentous bacteria appeared to initiate granule formation and generate the structures that supported further granule growth. It was observed that mature granules supported microbial populations that differed from nascent granules and were predominantly packed with coccoid bacteria. It was further observed that the diversity of the granular microbial community increased as the granules grew. Accumulibacter, Nitrosospira and Thauera were mainly responsible for nutrient removal while microorganisms such as Rhodocyclus and Hyphomicrobiaceae appeared to be primarily responsible for forming and maintaining the granule structure.     

Carbon,nitrogen and phosphorus removal mechanisms of aerobic granules

Aerobic granules are the potential tools to develop modern wastewater treatment technologies with improved nutrient removal efficiency. These granules have several promising advantages over conventional activated sludge-based wastewater treatment processes. This technology has the potential of reducing the infrastructure and operation costs of wastewater treatment by 25%, energy requirement by 30%, and space requirement by 75%. The nutrient removal mechanisms of aerobic granules are slightly different from that of the activated sludge. For instance, unlike activated sludge process, according to some reports, as high as 70% of the total phosphorus removed by aerobic granules were attributed to precipitation within the granules. Similarly, aerobic granule-based technology reduces the total amount of sludge produced during wastewater treatment. However, the reason behind this observation is unknown and it needs further explanations based on carbon and nitrogen removal mechanisms. Thus, as a part of the present review, a set of new hypotheses have been proposed to explain the peculiar nutrient removal mechanisms of the aerobic granules.     

Aerobic granular sludge: characterization,mechanism of granulation and application to wastewater treatment

Aerobic granular sludge can be classified as a type of self-immobilized microbial consortium, consisting mainly of aerobic and facultative bacteria and is distinct from anaerobic granular methanogenic sludge. Aerobic granular technology has been proposed as a promising technology for wastewater treatment, but is not yet established as a large-scale application. Aerobic granules have been cultured mainly in sequenced batch reactors (SBR) under hydraulic selection pressure. The factors influencing aerobic granulation, granulation mechanisms, microbial communities and the potential applications for the treatment of various wastewaters have been studied comprehensively on the laboratory-scale. Aerobic granular sludge has shown a potential for nitrogen removal, but is less competitive for the high strength organic wastewater treatments. This technology has been developed from the laboratory-scale to pilot scale applications, but with limited and unpublished full-scale applications for municipal wastewater treatment. The future needs and limitations for aerobic granular technology are discussed.