A comparative study on the formation and characterization of aerobic 4-chloroaniline-degrading granules in SBR and SABR

The formation and characterization of the aerobic 4-chloroaniline-degrading granules in the three column-type sequencing batch reactors were investigated in this paper. The granular sludge was observed since 15 days after start-up in R2 and R3 which had the high ratio of height to diameter (H/D). Since then and within the subsequent 75 days, the granulation of aerobic sludge was apparently developed by the decreased settling time and gradually increased 4-chloroaniline (4-ClA) concentration to above 400 mg.L(-1) in R1 to R3. The aerobic granules tended to be mature in all reactors continuously operated with 4-ClA loading rates of around 800 g.m(-3).d(-1), and the removal efficiencies of chemical oxygen demand, total nitrogen, and 4-ClA were maintained above 93%, 70%, and 99.9%, respectively. Mature aerobic granules in R1 to R3 featured with the average diameter of 0.78, 1.68, and 1.25 mm, minimal settling velocity of 20.5, 70.1 and 66.6 m.h(-1), specific 4-ClA degradation rates of 0.14, 0.21, and 0.27 g.gVSS(-1).d(-1), and the ratio of proteins to polysaccharides of 8.2, 10.8, and 13.7 mg.mg(-1), respectively. This study demonstrates that the reactor with a high H/D ratio and internal circulation favors the granulation and stabilization of aerobic sludge.     

Variable aeration in sequencing batch reactor with aerobic granular sludge

This study investigated the effects of reduced aeration in famine period on the performance of sequencing batch reactor (SBR) with aerobic granular sludge. Aerobic granules were first cultivated in two SBRs (R1 and R2) with acetate as sole carbon source. From operation day 27, aeration rate in R1 was reduced from 1.66 to 0.55 cm s(-1) from 110 min to the end of each cycle and further reduced from 30 min to the end of each cycle from day 63. R2 as a control was operated with a constant aeration rate of 1.66 cm s(-1) in the whole cycle during the entire experimental period. Results showed that changing trends of SVI, concentration, average size and VSS/SS of biomass with time in R1 and R2 were similar although different aeration modes were adopted. At steady state, SVI of aerobic granules and biomass concentration maintained at about 40 ml g(-1) and 6 g l(-1), respectively. Average size of granules was about 750 microm in R1 while 550 microm in R2. This is the first study to demonstrate that aerobic granular sludge could be stable at reduced aeration rate in famine period during more than 3-month operation. Such an operation strategy with reduced aeration rate will lead to a significant reduction of energy consumption, which makes the aerobic granular sludge technology more competitive over conventional activated sludge process. Furthermore, the stability of aerobic granular system with variable aeration further indicates that the difference of physiology and kinetics of aerobic granule in feast and famine periods results in the different requirements of oxygen and shear stress for the stability of granules, which will deepen the understanding of mechanism of aerobic granulation in sequencing batch reactor.     

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.     

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 effects of shear force on the formation, structure and metabolism of aerobic granules

The effect of shear force on aerobic granulation was studied in four column-type, sequential aerobic sludge blanket reactors. Hydrodynamic turbulence caused by upflow aeration served as the main shear force in the systems. Results showed that aerobic granulation was closely associated with the strength of shear force. Compact and regular aerobic granules were formed in the reactors with a superficial upflow air velocity higher than 1.2 cm s(-1). However, only typical bioflocs were observed in the reactor with a superficial upflow air velocity of 0.3 cm s(-1) during the whole experimental period. The characteristics of the aerobic granules in terms of settling ability, specific gravity, hydrophobicity, polysaccharide and protein content and specific oxygen utilization rate (SOUR) were examined. It was found that the shear force has a positive effect on the production of polysaccharide, SOUR, hydrophobicity of cell surface and specific gravity of granules. The hydrophobicity of granular sludge is much higher than that of bioflocs. Therefore, it appears that hydrophobicity could induce and further strengthen cell-cell interaction and might be the main force for the initiation of granulation. The shear-stimulated production of polysaccharides favors the formation of a stable granular structure. This research provides experimental evidence to show that shear force plays a crucial role in aerobic granulation and further influences the structure and metabolism of granules.     

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.     

降解苯胺和氯苯胺类污染物好氧污泥颗粒化及微生物种群结构分析

以序批式气提生物反应器(SABR)为平台,研究了苯胺和氯苯胺类有毒有机废水处理过程好氧污泥颗粒化。结果表明,通过缩短污泥沉降时间、逐步提升目标污染物进水负荷,反应器连续运行3个月,最终在污泥沉降时间5min、COD负荷1.0~3.6kg/(m3.d)、苯胺和氯苯胺负荷1kg/(m3.d)条件下实现污泥颗粒化,COD、苯胺和氯苯胺去除率分别稳定在90%、99.9%以上;获得的成熟好氧颗粒粒径在0.45~2.5mm,SOUR稳定在150mgDO/(gVSS·h)以上,颗粒污泥EPS中PN含量为28.0±1.9mg/gVSS,PN/PS比值为6.5mg/mg,苯胺类比降解速率达0.18g/(g·d);应用PCR-DGGE分子指纹图谱技术分析了稳定运行的颗粒化反应器内好氧污泥微生物种群结构,结果表明好氧颗粒内主要细菌分属β-Proteobacteria、γ-Proteobacteria及Flavobacteria等类群,优势菌为Pseudomonas sp.、Flavobacterium sp.;与已获得的降解氯苯胺好氧颗粒相比,苯胺存在下培养获得的好氧颗粒污泥微生物菌群结构更为丰富。     

好氧颗粒污泥对不同底物组成的响应

在序批式间歇反应器（R1、R2和R3）中,采用乙酸钠（R1）、蔗糖（R2）和苯酚（R3）三种不同基质作为碳源,均成功地培养出了好氧颗粒污泥;考察了不同颗粒污泥的理化性质及其对污染物的转化能力。结果表明,R1中颗粒污泥外观呈黄色,其主要的微生物菌群为细菌;R2中颗粒污泥外观呈黑色,内部含有丝状菌;而R3中颗粒污泥表面被大量丝状菌包裹,颗粒污泥呈淡黄色。在进水COD1000mg／L时R1、R2和R3中颗粒污泥比有机物的利用速率大小顺序为R3〉R1〉R2,而COD的去除率顺序却为R2〉R1〉R3。在进水氨氮40mg／L时,R1、R2和R3中氨氮的去除率分别在91％、96％和80％以上。以不同的底物培养出不同的好氧颗粒污泥可以拓展其在有毒化学物质如酚类化合物和高浓度工业废水生物处理中的应用。     

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.     

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.     

Effect of Mn<Superscript>2+</Superscript> augmentation on reinforcing aerobic sludge granulation in a sequencing batch reactor

Two sequencing batch reactors were synchronously operated to investigate the effect of manganese (II) (Mn²⁺) augmentation on aerobic granulation. Reactor 1 (R1) was added with 10 mg/L Mn²⁺, while there was no Mn²⁺ augmentation in reactor 2 (R2). Results showed that R1 had a faster granulation process than R2 and R1 performed better in chemical oxygen demand (COD) and ammonium nitrogen (NH₄⁺–N) removal efficiencies. Moreover, the mature granules augmented with Mn²⁺ behaved better on their physical characteristics and size distributions, and they also had higher production of extracellular polymeric substances (EPS) content. The result of three-dimensional excitation and emission matrix fluorescence showed that Mn²⁺ had the function of causing organic material diversity (especially proteins diversity) in EPS fraction from granules. Polymerase chain reaction and denaturing gradient gel electrophoresis techniques were employed to analyze the microbial and genetic characteristics in mature granules. The results exhibited that Mn²⁺ augmentation was mainly responsible for the higher microbial diversity of granules from R1 compared with that from R2. Uncultured sludge bacterium A16 (AF234726) and Rhodococcus sp. WTZ-R2 (HM004214) were the major species in R1, while only uncultured sludge bacterium A16 (AF234726) in R2. Moreover, there were eight species of organisms found in both two aerobic granules, and three species were found only in aerobic granules from R1. It could be concluded that Mn²⁺ could enhance the sludge granulation process and have a key effect role on the biological properties during the sludge granulation.     

Enhanced aerobic sludge granulation in sequencing batch reactor by Mg2+ augmentation

Two sequencing batch reactors (SBRs) were concurrently operated to investigate the effect of Mg(2+) augmentation on aerobic granulation. Augmentation with 10mg/l Mg(2+) in R2 significantly decreased the sludge granulation (defined as that over 15% of granules were larger than 0.6mm) time from 32 days to 18 days, at the same time, the mean diameter of the granules in R2 was 2.9 mm after the granulation, which was consistently larger than that (1.8mm) in R1. Mg(2+)-fed granules were denser and more compact, showed better settling and had higher polysaccharide contents, but it did not result in a difference in microbial morphology. The results demonstrated that Mg(2+) enhanced the sludge granulation process in the sequencing batch reactor.     

Understanding the properties of aerobic sludge granules as hydrogels

Aerobic sludge granules are larger, denser microbial aggregates than activated sludge flocs with a smoother and more regular surface, which facilitates greater wastewater treatment intensity. Factors important in their growth are still poorly understood, which is an impediment to the construction and operation of full-scale aerobic sludge granule processes. Data in this article obtained with granules treating an abattoir wastewater provide evidence that aerobic sludge granules are hydrogels. The results also demonstrate a method for characterizing macromolecular associations. The rheological profile of these granules was found to be analogous with that of typical polymer gels. Water uptake or swelling reflects an equilibrium between granule elastic modulus and osmotic pressure, whereby uptake is increased by reducing solute concentration or the elastic modulus. A weakening of the extracellular polymeric substance (EPS) matrix as demonstrated with mechanical spectroscopy was induced by several environmental factors including temperature, pH and ionic strength. Uniform and elastic deformation was observed at low strain. Enzymatic degradation studies indicate that proteins and alpha-polysaccharides were the major granule structural materials. The aerobic sludge granules in the current study were therefore protein-polysaccharide composite physical hydrogels. While aerobic sludge granules treating an abattoir wastewater are used as a case study, many of the fundamental principles detailed here are relevant to other granulation processes. The paradigm established in this study can potentially be applied to better understand the formation of aerobic sludge granules and thus overcome a hurdle in the acceptance of aerobic sludge granulation as an alternative to more traditional wastewater treatment processes.     

Enhanced aerobic granulation with extracellular polymeric substances (EPS)-free pellets

Extracellular polymeric substances (EPSs) were secreted by cells after they agglomerated into a compact aggregate. This study shows that the EPS initially embedded in seed sludge before granulation may sterically slow subsequent microbe–microbe contact, thereby delaying aerobic granulation. Three identical bioreactors were used in this study using glucose as the sole carbon and energy source. Reactor 1 (R1) was seeded with EPS-free pellets and operated in sequencing batch reactor (SBR) mode. Reactor 2 (R2) was seeded with the original sludge flocs and operated in SBR mode. Reactor 3 (R3) was seeded with EPS-free pellets and operated in continuously stirred tank reactor (CSTR) mode. Granulation occurred in R1 earlier than in R2; the granules that formed in R1 were larger and more compact than those in R2 at the same cultivation time. The few mature granules in R3 suggest that aerobic granulation can occur in a CSTR when a reactor is seeded with EPS-free pellets.     

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.     

Characteristics of aerobic granular sludge in a sequencing batch reactor with variable aeration

Aerobic granules can be used for the treatment of industrial or municipal wastewater, but high aeration rate is required for the stable operation of the granular sludge system. Therefore, the aim of this research was to reduce aeration rate greatly to decrease the energy consumption for the technology of aerobic granules. Based on the characteristics of sequencing batch reactor with distinct feast and famine periods, aeration rate was reduced from 1.66 to 0.55 cm s⁻¹ in the famine period after granules were formed. It was found that the settleability of aerobic granules in reactor R1 with reduced aeration was the same as that of aerobic granules in reactor R2 with constant aeration rate of 1.66 cm s⁻¹. However, the outer morphology of aerobic granules gradually changed from round shape to long shape, and minor population showed certain shift after aeration rate was reduced in the famine period. Since good settleability is the most essential feature of aerobic granules, it can be said that reducing aeration rate in famine period did not influence the stable operation of aerobic granular sludge system. Furthermore, the experimental results indicated that aeration rate in feast period was much more important to the stable operation of aerobic granules than that in famine period.     

A generalized model for settling velocity of aerobic granular sludge

Aerobic granulation is a novel biotechnology recently receiving intensive research attention. Aerobic granules developed in SBR can be as big as several millimeters, thus the traditional models describing the settling velocity of activated sludge are no long valid in aerobic granules culture. In this study, a new type of model was developed for the settling velocity of aerobic granules. This model shows that the settling velocity of aerobic granules is the function of SVI, mean size of granules and biomass concentration of granules. When the size of bioparticle is small enough, the proposed model reduces to the well-known Vesilind equation. Results indicated that the proposed model could satisfactorily fit experimental results obtained in the course of aerobic granulation under different conditions, while the Vesilind equation failed to or very poorly fit the experimental data. In addition, the proposed model can also be extended to anaerobic granules. The settling velocity is one of the most important parameters in both aerobic and anaerobic granulation, and successful biogranulation is highly related to the manipulation of settling velocity. It was demonstrated that the proposed model can sever as a useful tool for design and operation engineers to properly select the settling velocity for enhanced aerobic and anaerobic granulation.     

Aerobic granulation with brewery wastewater in a sequencing batch reactor

Aerobic granular sludge was cultivated in a sequencing batch reactor fed with brewery wastewater. After nine-week operation, stable granules with sizes of 2-7 mm were obtained. With the granulation, the SVI value decreased from 87.5 to 32 mL/g. The granular sludge had an excellent settling ability with the settling velocity over 91 m/h. Aerobic granular sludge exhibited good performance in the organics and nitrogen removal from brewery wastewater. After granulation, high and stable removal efficiencies of 88.7% COD(t), 88.9% NH(4)(+)-N were achieved at the volumetric exchange ratio of 50% and cycle duration of 6h. The average COD(t) and COD(s) of the effluent were 212 and 134 mg/L, respectively, and the average effluent ammonium concentration was less than 14.4 mg/L. Nitrogen was removed due to nitrification and simultaneous denitrification in the inner core of 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.     

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.