Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.     

Determination of the permeability and porosity of anaerobic sludge granules by size exclusion chromatography

Summary A new application of size-exclusion chromatography is described for assessment of the permeability and internal pore distribution of anaerobic sludge granules. The fractionation range and adsorption characteristics were investigated for a series of standard proteins and dextrans. To determine possible adsorption of solutes and stability of the sludges, the pH and salt concentration of the mobile phase were varied. Good results were obtained using dextrans as solutes and tap water as the mobile phase. To inhibit the sludge activity without affecting the granule characteristics the experimental arrangement was operated at 4°C. Three granular sludge types were investigated. The permeability of the granular sludges varied from 7% to 96%. The exclusion limit expressed as molecular mass also showed large differences. For two sludges, molecules greater than 80 000 Da cannot penetrate the pores; for one sludge the exclusion limit is 1300 Da. Experiments using acetic acid as an indicator of permeability gave corresponding results.Offprint requests to: P. A. Alphenaar     

Long-term impact of dissolved O(2) on the activity of anaerobic granules

The impact of influent dissolved O(2) on the characteristics of anaerobic granular sludge was investigated at various dissolved O(2) concentrations (0.5-8.1 ppm) in 1- and 5-L laboratory-scale upflow anaerobic sludge bed (UASB)-like anaerobic/aerobic coupled reactors with a synthetic wastewater (carbon sources containing 75% sucrose and 25% acetate). The rate of dissolved O(2) supplied to the coupled reactor was as high as 0.40 g O(2)/L(rx).d, and the anaerobic/aerobic coupled reactors maintained excellent methanogenic performances at a COD loading rate of 3 g COD/L(rx).d even after the reactors had been operated with dissolved O(2) for 3 months. The activities of granular sludge on various substrates (glucose, propionate, and hydrogen) were not impaired, and acetate activity was even improved over a short term. However, after 3 months of operation, slight declines on the acetoclastic activities of granules were observed in the coupled reactor receiving the recirculated fluid containing 8.1 ppm dissolved O(2).Methane yield in the anaerobic control reactor and anaerobic/aerobic coupled reactors revealed that a significant aerobic elimination (up to 30%) of substrate occurred in the coupled reactors, as expected. The presence of dissolved O(2) in the recirculated fluid resulted in the development of fluffy biolayers on the granule surface, which imposed a negative impact on the settleability of granular sludge and caused a slightly higher sludge washout. This research shows that the anaerobic/aerobic coupled reactor can be successfully operated under O(2)-limited conditions and is an ideal engineered ecosystem integrating oxic and anaerobic niches. (c) 1996 John Wiley & Sons, Inc.     

Influence of substrate surface loading on the kinetic behaviour of aerobic granules

In the aerobic granular sludge reactor, the substrate loading is related to the size of the aerobic granules cultivated. This study investigated the influence of substrate surface loading on the growth and substrate-utilization kinetics of aerobic granules. Results showed that microbial surface growth rate and surface biodegradation rate are fairly related to the substrate surface loading by the Monod-type equation. In this study, both the theoretical maximum growth yield and the Pirt maintenance coefficient were determined. It was found that the estimated theoretical maximum growth yield of aerobic granules was as low as 0.2 g biomass g^–1 chemical oxygen demand (COD) and 10–40% of input substrate-COD was consumed through the maintenance metabolism, while experimental results further showed that the unit oxygen uptake by aerobic granules was 0.68 g oxygen g^–1 COD, which was much higher than that reported in activated sludge processes. Based on the growth yield and unit oxygen uptake determined, an oxidative assimilation equation of acetate-fed aerobic granules was derived; and this was confirmed by respirometric tests. In aerobic granular culture, about 74% of the input substrate-carbon was converted to carbon dioxide. The growth yield of aerobic granules was three times lower than that of activated sludge. It is likely that high carbon dioxide production is the main cause of the low growth yield of aerobic granules, indicating a possible energy uncoupling in aerobic granular culture.     

Bioaugmentation and enhanced formation of microbial granules used in aerobic wastewater treatment

Microbial aggregates of an aerobic granular sludge can be used for the treatment of industrial or municipal wastewater, but their formation from a microbial activated sludge requires several weeks. Therefore, the aim of this research was the selection of microbial cultures to shorten the granule-forming period from several weeks to a few days. An enrichment culture with the ability to accelerate granulation was obtained by repeating the selection and batch cultivation of fast-settling microbial aggregates isolated from the aerobic granular sludge. Bacterial cultures of Klebsiella pneumoniae strain B and Pseudomonas veronii strain F, with self-aggregation indexes of 65 and 51%, respectively, and a coaggregation index of 58%, were isolated from the enrichment culture. A mixture of these strains with the activated sludge was used as an inoculum in an experimental sequencing batch reactor to start up an aerobic granulation process. Aerobic granules with a mean diameter of 446±76 μm were formed in an experiment after 8 days of cultivation, but microbial granules were absent in controls. Considering biosafety issues, K. pneumoniae strain B was excluded from further studies, but P. veronii strain F was selected for larger-scale testing.Stephen Tiong-Lee Tay Passed away on 27 July 2005.     

Starvation is not a prerequisite for the formation of aerobic granules

Activated sludge with sludge volume index (SVI)₃₀ of 77 ml g⁻¹ and SVI₃₀ of 433 ml g⁻¹ was inoculated to start up reactors R1 and R2, respectively. In both R1 and R2, cycle time of 1 h and the influent chemical oxygen demand (COD) concentrations of 1,000 mg l⁻¹ were employed. Initial settling time of 2 min resulted in the loss of a substantial amount of biomass as wash-out and high effluent COD concentrations within the first week of operation. This implied that there was no starvation phase in each cycle of R1 and R2 during the first week of operation. However, aerobic granules with a size above 400 μm formed by day 7. Thus, it was concluded that starvation was not a prerequisite for the formation of aerobic granules. When cycle time was 1 h, the instability of aerobic granules was observed. When cycle time was prolonged to 1.5 h and granular sludge of 200 ml was used to start up reactor R3, the reactor R3 reached steady state within 1 week. SVI, size, and the morphology of granular sludge in R3 remained stable during the 47-day operation, which indicated that prolonged starvation time had positive effects on the stability of aerobic granules.     

Microbial community in granules from a high-rate EGSB reactor

The spatial distribution, quantity and diversity of different microorganisms within anaerobic granular sludge from a lab-scale expanded granular sludge bed (EGSB) reactor operated at different organic loading rates were studied using florescent in situ hybridization (FISH), real time quantitative — polymerase chain reaction (RTQ-PCR) and denaturing gradient gel electrophoresis (DGGE) techniques. The results indicated that most Eubacteria were located in the outer layer of granule, while the Archaea which mainly were methanogens and more sensible to the environmental conditions were located in the inner layer of the granule. The quantity of Archaea was obviously less than that of Eubacteria in the granules, but increased with the increasing of organic loading rates of the reactor. As the organic loading rate of the reactor increased and the operating time elapsed, the Archaea community in the granules changed significantly. Seven typical DGGE bands were collected and sequenced, and found that the dominant species of Archaea in the granules operated in the last period were mainly Methanocorpusculum, Methanobacterium, Methanosaeta.     

Different types of sludge granules in UASB reactors treating acidified wastewaters

The influence of a high energy substrate, i.e. sucrose, on the granular sludge yield and the development of different types of granular sludge was investigated by using Upflow Anaerobic Sludge Bed (UASB) reactors fed with synthetic wastewater. The feed COD was a mixture of volatile fatty acids (VFA) i.e., 20, 40, and 40% of the COD as C₂-, C₃-, and C₄-VFA, respectively. Furthermore, experiments were carried out in which 10 and 30% of the VFA COD was substituted with sucrose. The following distinctly different types of granules were observed in each testrun: in the reactor fed with solely VFA, black (B) and white (W) granules developed; in the reactor fed with a mixture of 90% VFA and 10% sucrose, three types of granules i.e., B, W, and grey (G) granules could be seen; in the reactor fed with 70% VFA and 30% sucrose, only W and G granules were found. The granular sludge yield increased proportional to the amount of sucrose COD. At steady-state performance of the reactors, specific acidogenic (SAA) and methanogenic (SMA) activity tests on these granules revealed that B granules had the highest SMA with low SAA. The W granules had very high SMA with low SAA. G granules gave the highest SAA with a considerable SMA. Measurement of coenzyme F₄₂₀ revealed that B granules consist mainly of acetoclastic methanogens. The fore-mentioned tests were supplemented with analyses of the wash-out cells present in the reactor effluent and the results suggested that acidogens, if present, prevail at the granule surface. The B granules were particularly rich in Ca, Mn, and Zn minerals. The size distribution analysis showed that the granule diameter increased in the following order: B相似文献   

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.     

Stability of aerobic granules during long-term bioreactor operation

Aerobic granular sludge technology has been extensively studied over the past 20 years and is regarded as the upcoming new standard for biological treatment of domestic and industrial wastewaters. Aerobic granules (AG) are dense, compact, self-immobilized microbial aggregates that allow better sludge-water separation and thereby higher biomass concentrations in the bioreactor than conventional activated sludge aggregates. This brings potential practical advantages in terms of investment cost, energy consumption and footprint. Yet, despite the relevant advances regarding the process of AG formation, instability of AG during long-term operation is still seen as a major barrier for a broad practical application of this technology. This paper presents an up-to-date review of the literature focusing on AG stability, aiming to contribute to the identification of key factors for promoting long-term stability of AG and to a better understanding of the underlying mechanisms. Operational conditions leading to AG disintegration are described, including high organic loads, particulate substrates in the influent, toxic feed components, aerobic feeding and too short famine periods. These operational and influent wastewater composition conditions were shown to influence the micro-environment of AG, consequently affecting their stability. Granule stability is generally favored by the presence of a dense core, with microbial growth throughout the AG depth being a crucial intrinsic factor determining its structural integrity. Accordingly, possible practical solutions to improve granule long-term stability are described, namely through the promotion of minimal substrate concentration gradients and control of microbial growth rates within AG, including anaerobic, plug-flow feeding and specific sludge removal strategies.     

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.     

Structural analysis of anaerobic granules in a phase separated reactor by electron microscopy

This paper discusses the microbial community structure of anaerobic granules and the effect of phase separation in anaerobic reactor on the characteristics of granules. Electron micrographs revealed that the core of anaerobic granular sludge consists predominantly of Methanosaeta-like cells, a key microorganism in granulation process. Granules in the methanogenic dominant zone of the reactor were stable and densely packed with smooth regular surface. On the other hand, granules subjected to acidogenic activities were less stable structures with broken parts and an irregular fissured surface. Anaerobic granules consisted of a vast diversity of species from the outer surface to the core of the granule and possessed a multi-layered structure. Viruses in the granules suggests the presence of bacteriophage in the granular biomass. These could be responsible for destroying cells and weakening the internal structure of granules, and thus possibly causing the breaking of granules. The observation of protozoa-like microorganism on the exterior zone of granular structure is believed to play an important role as bacterial predator and control the growth of bacterial cells. The images observed in this study shows that anaerobic granule harbour diverse number of microbial species, and act differently in acidogenic and methanogenic microbial zones.     

厌氧反应器中颗粒污泥的培育及应用研究进展*

厌氧生物处理技术因其具有有机负荷高、污泥产量低、能耗低等优点被广泛应用于各种废水处理中。厌氧颗粒污泥具有沉降性能好、微生物浓度高、有机负荷高等优点,极大地提高了废水处理效率。尤其在处理含高氨氮废水中,厌氧颗粒污泥的形成对反应器的高效生物脱氮至关重要。但到目前为止,厌氧反应器中的颗粒污泥形成及废水处理效果还缺乏系统的认识。鉴于此,总结了厌氧反应器中颗粒污泥的形成机制,分析了影响厌氧反应器中颗粒污泥形成的因素,论述了厌氧反应器中厌氧颗粒污泥生长的模拟,最后介绍了厌氧颗粒污泥在国内外的主流应用。厌氧反应器中颗粒污泥的形成是综合因素影响的结果,对影响厌氧颗粒污泥形成的每个因素都需要认真对待,可为在厌氧反应器中颗粒污泥的培育和应用提供理论指导和技术支撑。     

Novel technique for measuring the size distribution of granules from anaerobic reactors for wastewater treatment

A new technique for measuring the size distribution of anaerobic granular sludge, which involves the use of digital image analysis, is presented. Sludge samples are embedded in gelatin, spread over glass dishes, which are then placed over a flat-bed scanner where an image is captured. The images are processed with an image analysis software. The technique is simple, reliable and does not need any special equipment. © Rapid Science Ltd. 1998     

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.     

Stoichiometric analysis of dissolved organic carbon flux into storage and growth in aerobic granules culture

This study attempted to address a fundamental question of whether metabolic behaviors of aerobic granules are different from their counterparts, such as activated sludge and biofilms. A series of respirometric experiments were carried out using mature aerobic granules with mean sizes of 0.75–3.4 mm. Results suggested that metabolism of aerobic granules comprised three consecutive phases: (i) conversion of external dissolved organic carbon to a poly-β-hydroxybutyrate-like substance; (ii) growth of aerobic granules on the stored poly-β-hydroxybutyrate-like substance derived from phase I, and (iii) subsequent endogenous metabolism of aerobic granules. The stoichiometric analysis revealed that the conversion yields of external dissolved organic carbon to the poly-β-hydroxybutyrate-like substance, the growth yields of biomass on storage, and the overall growth yields of biomass on external dissolved organic carbon were not significantly correlated to the sizes of aerobic granules, i.e., the metabolism of aerobic granules would be size independent. The conversion coefficients and growth rates of aerobic granules were found to be comparable with those reported in the activated sludge and biofilms cultures, indicating that there would not be significant difference in the metabolisms of aerobic granules over activated sludge and biofilms. This information will be useful for modeling and designing aerobic granular sludge processes.     

Fast start-up, performance and microbial community in a pilot-scale anammox reactor seeded with exotic mature granules

The possibility to introduce the exotic anammox sludge to seed the pilot-scale anammox granular reactor and its fast start-up for treating high nitrogen concentration wastewater were evaluated in this study. The reactor was started up successfully in two weeks; in addition, high nitrogen removal was achieved for a long period. Stoichiometry molar ratios of nitrite conversion and nitrate production to ammonium conversion were calculated to be 1.26 ± 0.02:1 and 0.26 ± 0.01:1, respectively. The Stover-Kincannon model which was first applied in granular anammox process indicated that the granular anammox reactor possessed high nitrogen removal potential of 27.8 kg/m³/d. The anammox granules in the reactor were characterized via microscope observation and fluorescence in situ hybridization technique. Moreover, the microbial community of the granules was quantified to be composed of 91.4-92.4% anammox bacteria by real-time polymerase chain reaction. This pilot study can elucidate further information for industrial granular anammox application.     

Dispersal and control of anammox granular sludge at high substrate concentrations

This paper reports about the dispersal and control of anammox granular sludge at high substrate concentrations. The results demonstrate that anammox granular sludge would turn into flocculent sludge when the substrate concentrations exceed the inhibitory threshold concentrations, with an apparent drop in the settling velocity of anammox sludge from 73.73 to 16.49 m/h. Moreover, the sludge was washed out of the reactor and a decrease in the nitrogen removal rate from 23.82 to 16.97 kg N/(m³/day) was observed. The dominant anammox bacteria in the granular and flocculent sludge were Candidatus Kuenenia stuttgartiensis; however, the contents of heme c and extracellular polymeric substances in the flocculent sludge were much lower than in the granular sludge. Furthermore, the chemical composition of extracellular polymeric substances was different. The high nitrite concentrations more than the inhibitory threshold concentrations were regarded as the reason for the observed granular sludge dispersal and deterioration in reactor performance. The apparent dispersed granular sludge and malfunction of reactor performance could be recovered by means of washing out the residual substrate from the reactor and then re-running the reactor from low substrate concentrations.     

Cultivation of low-temperature (15°C), anaerobic, wastewater treatment granules

Aims: Anaerobic sludge granules underpin high‐rate waste‐to‐energy bioreactors. Granulation is a microbiological phenomenon involving the self‐immobilization of several trophic groups. Low‐temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low‐temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low‐temperature (R2 and R3, 15°C) laboratory‐scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose‐based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low‐temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H₂/CO₂‐mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high‐rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented.     

高活性厌氧颗粒污泥稳定性及基质代谢和种间氢转移的研究

选择了几种废水形成的厌氧污泥,进行了稳定性、基质代谢及种间氢转移的研究.颗粒化的污泥对盐、pH、酶作用、温度、压力等外界条件影响有一定的抗性,在丙酸代谢中,丙酸对颗粒污泥抑制浓度可达1000mg/L,而絮状污泥在500mg/L就被明显抑制,并比较了两者的最大比产甲烷速率和氢酶活性,在种间氢转移实验中,乙醇对颗粒污泥的抑制浓度比絮状污泥要高,颗粒污泥在各方面的性能都明显优于絮状污泥,还对颗粒污泥的物理特性及保存方式进行了初步研究.