Relationship between size and mass transfer resistance in aerobic granules

AIMS: To investigate the size effect of aerobic granules on mass transfer efficiency by introducing the effective factor and the modified Thiele modulus. METHODS AND RESULTS: Batch experiments of aerobic granules with different sizes were conducted to study the size effect of granules on mass transfer resistance. Results showed that both specific substrate removal and biomass growth rates were size dependent, i.e. reduced rates were observed at big sizes. It was found that the diffusion resistance described by the effective factor and the Thiele modulus increased with the increase of the size of aerobic granules. CONCLUSIONS: The effective factor should be controlled at values higher than 0.44 and the Thiele modulus lower than 1.05 for efficient mass transfer in aerobic granules. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the coupled effective factor and Thiele modulus, an operation guidance including granule radius, kinetics of biomass and environmental conditions could be proposed for stable aerobic granulation.     

Growth and storage processes in aerobic granules grown on soybean wastewater

Aerobic granules in a sequencing batch reactor (SBR) are subjected to alternative feast and famine conditions, and are able to take up carbon substrate in wastewater rapidly and to store it as intracellular storage products when the substrate is in excess. This phenomenon could not be described by the widely used activated sludge model No.3 (ASM3). In this work, taking adsorption process, microbial maintenance, and substrate diffusion into account, the simultaneous growth and storage processes occurring in an aerobic-granule-based SBR are investigated with experimental and modeling approaches. A new model is established and successfully validated with the experimental results of an SBR fed with soybean-processing wastewater. Simulation results show that our approach is appropriate for elucidating the fates of major model components. Comparison between ASM3 and the model established in this work demonstrates that the latter is better to describe the substrate removal mechanisms and simultaneous growth and storage processes in aerobic granules.     

Degradation of phenol by aerobic granules and isolated yeast Candida tropicalis

Aerobic granules effectively degrade phenol at high concentrations. This work cultivated aerobic granules that can degrade phenol at a constant rate of 49 mg-phenol/g x VSS/h up to 1,000 mg/L of phenol. Fluorescent staining and confocal laser scanning microscopy (CLSM) tests demonstrated that an active biomass was accumulated at the granule outer layer. A strain with maximum ability to degrade phenol and a high tolerance to phenol toxicity isolated from the granules was identified as Candida tropicalis via 18S rRNA sequencing. This strain degrades phenol at a maximum rate of 390 mg-phenol/g x VSS/h at pH 6 and 30 degrees C, whereas inhibitory effects existed at concentrations >1,000 mg/L. The Haldane kinetic model elucidates the growth and phenol biodegradation kinetics of the C. tropicalis. The fluorescence in situ hybridization (FISH) and CLSM test suggested that the Candida strain was primarily distributed throughout the surface layer of granule; hence, achieving a near constant reaction rate over a wide range of phenol concentration. The mass transfer barrier provided by granule matrix did not determine the reaction rates for the present phenol-degrading granule.     

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.     

Hydraulic characteristics of aerobic granules using size exclusion chromatography

Elution of poly(ethylene glycol) of molecular weight 200-20,000 Da from a size exclusion chromatography column packed with phenol-fed aerobic granules of three different nominal sizes (types I-III) has been investigated. The pore sizes of the three types of granules were evaluated based on the mean hydraulic times of the elution curves that decreased directly proportional to the increased logarithm of the molecular mass of a standard tracer and increased as granule size decreased. The corresponding exclusion limits for types I-III granules were 139,000, 123,000, and 54,500 Da, respectively. A one-dimensional convection-dispersion model described the effective dispersion coefficients of the tracers through the granule column. The intra-granular permeabilities and convective and diffusional transit times through the granule interior were evaluated by a dual porosity model. For small molecules of molecular mass <5,000 Da, intra-granular convection dominated transport mechanisms at fast moving velocity. For comparatively larger molecules, diffusion barrier existed to limit nutrient supply to the granules. The size exclusion test provided intra granular transport characteristics using detailed analysis on the elution data.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

Mechanism of calcium accumulation in acetate-fed aerobic granule

High calcium content has been widely reported in acetate-fed aerobic granules, but the reason behind this is unclear yet. By SEM–energy dispersive X-ray mapping analysis, this study showed that the majority of calcium was presented in the central part of the acetate-fed aerobic granule, and the granule shell part was nearly calcium-free. The elemental analysis of calcium ions coupled with the chemical titration of carbonate further revealed that the calcium ions that accumulated in the acetate-fed aerobic granule mainly existed in the form of calcium carbonate (CaCO₃). The formation of the CaCO₃ appeared to be highly dependent on the size of the aerobic granule, i.e., the CaCO₃ precipitation was found only in aerobic granules with radiuses larger than 0.5 mm. These experimental observations with regard to the formation of CaCO₃ in the acetate-fed aerobic granule were further confirmed by the model simulation, which was based on the principles of mass diffusion and carbonate dissociation in liquid phase. This study for the first time showed that the size of the acetate-fed aerobic granule would indeed play an essential role in the CaCO₃ formation, and provided experimental evidence that a crystal CaCO₃ core was not necessarily required for granulation.     

Amylase activity in substrate deficiency aerobic granules

Immunohistochemical staining was applied together with the multicolor fluorescent scheme to demonstrate the amylase activity for polysaccharide hydrolysis in stored or starved aerobic granules that are in substrate deficiency. If sufficient nutrients were present, α-amylase and β-amylase were found close to the surface layer of the original granules. Following storage or starvation during which most external nutrients were depleted, the α-amylase and β-amylase were distributed over the entire granule interior, suggesting endogenous respiration at the core of the granule. In particular, the fluorescent intensities of α-amylase and β-amylase were enriched 5–20 μm from the edge of the internal cavity, suggesting the strong correlation between polysaccharide hydrolysis and the formation of interior cavities. The secreted amylase was located near the living cells, suggesting that the polysaccharide hydrolysis is restricted to local environment that occurs near the functional strains. Internal hydrolysis within the core, for the case of both proteins and polysaccharides should correspond in principle to the loss of granule stability.     

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。     

The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses

Aerobic sewage granules are dense, spherical biofilms, regarded as a useful and promising tool in wastewater treatment processes. Recent studies revealed that fungi can be implemented in biofilm formation. This study attempts to uncover the fungal diversity in aerobic granules by sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer regions. For this purpose, appropriate PCR and sequencing primer sets were selected and an improved DNA isolation protocol was used. The sequences of 41 isolates were assigned to the taxonomic groups Pleosporaceae, Xylariales, Theleobolaceae, Claviceps, Aureobasidium, Candida boleticola , and Tremellomycetes within the fungi. It turned out that the fungal community composition in granules depended on the wastewater type and the phase of granule development.     

Distribution of extracellular polymeric substances in aerobic granules

Extracellular matrix provides an architectural structure and mechanical stability for aerobic granules. Distributions of cells and extracellular polymeric substances (EPS), including proteins, α- and β-d-glucopyranose polysaccharides, in acetate-fed granules and phenol-fed granules were probed using a novel quadruple staining scheme. In acetate-fed granules, protein and β-d-glucopyranose polysaccharides formed the core, whereas, the cells and α-d-glucopyranose polysaccharides accumulated in the granule outer layers. Based on these experimental findings, this study indicated that different conclusions can be obtained regarding EPS distributions when granules were stained differently. The core of phenol-fed granules, conversely, was formed principally by proteins; whereas, the cells and α- and β-d-glucopyranose polysaccharides were accumulated at an outer filamentous layer. Using a series of confocal laser scanning microscope (CLSM) images whose threshold values were determined via Otsu’s scheme, the three-dimensional distributions of cells and EPS were produced using a polygonal surface model. Structural information extracted can be applied in further development of comprehensive granule models.     

Effective diffusivity of oxygen in microbial pellets

In a typical submerged aerobic fermentation with microbial pellets, the effective diffusivity of oxygen in the pellets is probably the most important, yet most difficult transport property to characterize experimentally. Its values directly indicate the efficiency or deficiency of oxygen to individual cells, and thus the biological activity of the microorganisms. In the past, it was not possible to assess reliably the effective diffusivity of oxygen in pellets due to several reasons. Firstly, most oxygen electrodes available were coarse, and hence not suitable for in situ measurements. Secondly, there was a lack of methods rigorous enough to characterize the structure of the microbial pellets. A state-of-the-art review of the literature relating to the feature subject is presented. Emphasis is laid upon development and evolution of the means for quantitative characterization of the effective diffusivity of oxygen in microbial pellets.     

好氧反硝化菌的研究进展

综述了好氧反硝化菌的种类和特性、好氧反硝化菌的反硝化作用机制和影响因素.好氧反硝化菌主要包括假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)、副球菌属(Para-coccus)和芽孢杆菌属(Bacillus)等,属好氧或兼性好氧异养微生物.好氧反硝化菌能在好氧条件下进行反硝化,其主要产物是N2O,并可将铵态氮直接转化成气态产物.催化好氧反硝化菌反硝化作用的硝酸盐还原酶是周质酶而不是膜结合酶.溶解氧和C/N往往是影响好氧反硝化菌反硝化作用的主要因素.介绍了间歇曝气法、选择性培养基法等好氧反硝化菌的主要分离筛选方法.概述了好氧反硝化菌在水产养殖、废水生物处理、降解有机污染物以及对土壤氮素损失的影响方面的研究进展.     

Storage and growth of denitrifiers in aerobic granules: part II. model calibration and verification

A mathematical model to describe the simultaneous storage and growth activities of denitrifiers in aerobic granules under anoxic conditions has been developed in an accompanying article. The sensitivity of the nitrate uptake rate (NUR) toward the stoichiometric and kinetic coefficients is analyzed in this article. The model parameter values are estimated by minimizing the sum of squares of the deviations between the measured and model-predicted values. The model is successfully calibrated and a set of stoichiometric and kinetic parameters for the anoxic storage and growth of the denitrifiers are obtained. Thereafter, the model established is verified with three set of experimental data. The comparison between the model established with the ASM1 model and ASM3 shows that the present model is appropriate to simulate and predict the performance of a granule-based denitrification system.     

Biodegradability of extracellular polymeric substances produced by aerobic granules

This study investigated the biodegradability of extracellular polymeric substances (EPS) produced by aerobic granules. Aerobic granules were precultivated with synthetic wastewater in a lab-scale sequencing batch reactor. EPS were extracted from aerobic granules and were then fed as the sole carbon source to their own producers. Results showed that about 50% of EPS produced by aerobic granules could be utilized by their producers under aerobic starvation condition. The average biodegradation rate of the granule EPS in terms of chemical oxygen demand was five times slower than that of acetate, but 50 times faster than that of nonbiodegradable EPS produced by aerobic granules. The nonbiodegradable EPS was mainly found on the outer shell of aerobic granule. EPS produced by aerobic granules basically comprised two major components, i.e., biodegradable and nonbiodegradable EPS. The biodegradable EPS could serve as a useful energy source to sustain the growth of aerobic granules under starvation. This study provides experimental evidence that part of the EPS produced by aerobic granules would be biodegradable, but only nonbiodegradable EPS would play a crucial role in maintaining the structural integrity of aerobic granule.     

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.     

Storage and growth of denitrifiers in aerobic granules: part I. model development

A mathematical model, based on the Activated Sludge Model No.3 (ASM3), is developed to describe the storage and growth activities of denitrifiers in aerobic granules under anoxic conditions. In this model, mass transfer, hydrolysis, simultaneous anoxic storage and growth, anoxic maintenance, and endogenous decay are all taken into account. The model established is implemented in the well-established AQUASIM simulation software. A combination of completely mixed reactor and biofilm reactor compartments provided by AQUASIM is used to simulate the mass transport and conversion processes occurring in both bulk liquid and granules. The modeling results explicitly show that the external substrate is immediately utilized for storage and growth at feast phase. More external substrates are diverted to storage process than the primary biomass production process. The model simulation indicates that the nitrate utilization rate (NUR) of granules-based denitrification process includes four linear phases of nitrate reduction. Furthermore, the methodology for determining the most important parameter in this model, that is, anoxic reduction factor, is established.     

Heterogeneous diffusion in aerobic granular sludge

Aerobic granular sludge (AGS) technology allows simultaneous nitrogen, phosphorus, and carbon removal in compact wastewater treatment processes. To operate, design, and model AGS reactors, it is essential to properly understand the diffusive transport within the granules. In this study, diffusive mass transfer within full-scale and lab-scale AGS was characterized with nuclear magnetic resonance (NMR) methods. Self-diffusion coefficients of water inside the granules were determined with pulsed-field gradient NMR, while the granule structure was visualized with NMR imaging. A reaction-diffusion granule-scale model was set up to evaluate the impact of heterogeneous diffusion on granule performance. The self-diffusion coefficient of water in AGS was ∼70% of the self-diffusion coefficient of free water. There was no significant difference between self-diffusion in AGS from full-scale treatment plants and from lab-scale reactors. The results of the model showed that diffusional heterogeneity did not lead to a major change of flux into the granule (<1%). This study shows that differences between granular sludges and heterogeneity within granules have little impact on the kinetic properties of AGS. Thus, a relatively simple approach is sufficient to describe mass transport by diffusion into the granules.     

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.     

Oxygen diffusion and consumption in active aerobic granules of heterogeneous structure

The interior structure of aerobic granules is highly heterogeneous, hence, affecting the transport and reaction processes in the granules. The granule structure and the dissolved oxygen profiles were probed at the same granule in the current work for possible estimation of transport and kinetic parameters in the granule. With the tested granules fed by phenol or acetate as carbon source, most inflow oxygen was consumed by an active layer thickness of less than 125 μm on the granule surface. The confocal laser scanning microscopy scans also revealed a surface layer thickness of approximately 100 μm consisting of cells. The diffusivities of oxygen transport and the kinetic constant of oxygen consumption in the active layers only were evaluated. The theoretical models adopted in literature that ignored the contributions of the layered structure of aerobic granule could have overlooked the possible limitations on oxygen transport.