Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate reduction in methanol and formate-fed UASB reactors

The feasibility of thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate-reducing processes was investigated in three lab-scale upflow anaerobic sludge bed (UASB) reactors fed with either methanol or formate as the sole substrates and inoculated with mesophilic granular sludge previously not exposed to high temperatures. Full methanol and formate degradation at temperatures up to, respectively, 70 and 75 degrees C, were achieved when operating UASB reactors fed with sulfate rich (COD/SO4(2-)=0.5) synthetic wastewater. Methane-producing archaea (MPA) outcompeted sulfate-reducing bacteria (SRB) in the formate-fed UASB reactor at all temperatures tested (65-75 degrees C). In contrast, SRB outcompeted MPA in methanol-fed UASB reactors at temperatures equal to or exceeding 65 degrees C, whereas strong competition between SRB and MPA was observed in these reactors at 55 degrees C. A short-term (5 days) temperature increase from 55 to 65 degrees C was an effective strategy to suppress methanogenesis in methanol-fed sulfidogenic UASB reactors operated at 55 degrees C. Methanol was found to be a suitable electron donor for sulfate-reducing processes at a maximal temperature of 70 degrees C, with sulfide as the sole mineralization product of methanol degradation at that temperature.     

Granular sludge formation in upflow anaerobic sludge blanket (UASB) reactors

The state of the art for upflow anaerobic sludge blanket (UASB) reactors is discussed, focusing on the microbiology of immobilized anaerobic bacteria and the mechanism of granule formation. The development of granular sludge is the key factor for successful operation of the UASB reactors. Criteria for determining if granular sludge has developed in a UASB reactor is given based on the densities and diameters of the granular sludge. The shape and composition of granular sludge can vary significantly. Granules typically have a spherical form with a diameter from 0.14 to 5 mm. The inorganic mineral content varies from 10 to 90% of the dry weight of the granules, depending on the wastewater composition etc. The main components of the ash are calcium, potassium, and iron. The extracellular polymers in the granular sludge are important for the structure and maintenance of granules, while the inorganic composition seems to be of less importance. The extracellular polymer content varies between 0.6 and 20% of the volatile suspended solids and consists mainly of protein and polysaccharides. Both Methanosaeta spp. (formerly Methanothrix) and Methanosarcina spp. have been identified as important aceticlastic methanogens for the initial granulation and development of granular sludge. Immunological methods have been used to identify other methanogens in the granules. The results have showed that, besides the aceticlastic methanogens Methanosaeta spp. and Methanosarcina spp., hydrogen and formate utilizing bacteria are also present, e.g., Methanobacterium formicicum, Methanobacterium thermoautotrophicum, and Methanobrevibacter spp. Microcolonies of syntrophic bacteria are often observed in the granules, and the significant electron transfer in these microcolonies occurs through interspecies hydrogen transfer. The internal organization of the various groups of bacteria in the granules depends on the wastewater composition and the dominating metabolic pathways in the granules. Internal organization is observed in granules where such an arrangement is beneficial for an optimal degradation of the wastewater. A four-step model is given for the initial development of granular sludge. (c) 1996 John Wiley & Sons, Inc.     

Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging to Chloroflexi Subphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55°C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Influence of biogas-induced mixing on granulation in UASB reactors

Studies have been carried out to correlate biogas-induced mixing and granulation in upflow anaerobic sludge blanket (UASB) reactors, treating low-strength as well as high-strength biodegradable wastewaters. A dimensionless granulation index (GI) has been framed taking into account the mixing in sludge bed due to produced biogas. Analysis of full-scale, pilot-scale and lab-scale UASB reactors treating actual wastewaters reveals the significance of biogas-induced mixing, represented by GI, on granulation of biomass in the reactors. For obtaining proper granulation in UASB reactors (percentage granules greater than 50%, w/w), resulting in higher chemical oxygen demand (COD) removal efficiency, it is recommended to maintain GI values in the range of 15,000–57,000.     

Anaerobic treatment of poultry mortality in a temperature-phased leachbed-UASB system

Anaerobic digestion has been proposed as an alternative to the conventional disposal methods of burial, incineration, rendering and aerobic composting. A temperature-phased system consisting of one UASB (at 55 degrees C) and three leach-bed reactors (at ambient temperatures) was tested for its efficiencies in treating poultry mortality. The thermophilic UASB was difficult to start-up. It also showed signs of inhibited methanogenesis. Chemical parameters such as long chain fatty acids, volatile fatty acids and ammonia concentrations were all very high for the thermophilic UASB. Lowering its temperature to 35 degrees C enhanced its stability and improved its performances. Lowering the pH of the 55 degrees C UASB also improved its chemical oxygen demand (COD) reduction efficiency as well as its methane production rate. The results were compared to that of another similar system where the UASB reactor was maintained at 35 degrees C instead of at 55 degrees C.     

Introduction of a de novo bioremediation ability, aryl reductive dechlorination, into anaerobic granular sludge by inoculation of sludge with Desulfomonile tiedjei.

Methanogenic upflow anaerobic granular-sludge blanket (UASB) reactors treat wastewaters at a high rate while simultaneously producing a useful product, methane; however, recalcitrant environmental pollutants may not be degraded. To impart 3-chlorobenzoate (3-CB)-dechlorinating ability to UASB reactors, we inoculated granular sludge in UASB reactors with either a pure culture of Desulfomonile tiedjei (a 3-CB-dechlorinating anaerobe) or a three-member consortium consisting of D. tiejei, a benzoate degrader, and an H2-utilizing methanogen. No degradation occurred in an uninoculated control reactor which was started with the same granular sludge, but inoculated reactors and granules from the inoculated UASB systems rapidly transformed 3-CB (54 mumol/day/g of granule biomass). After several months at a hydraulic retention time of 0.5 day, much shorter than the generation time of D. tiedjei, the reactors still dechlorinated 3-CB. This indicated that the bacteria were immobilized in the reactor granules, and by using an antibody probe for D. tiedjei, we demonstrated that this microorganism had colonized the sludge granules. These results represent the first addition of a pure culture or a defined microbial mixture to a viable waste treatment process to introduce a specific de novo degradative pathway into a granular-sludge consortium.     

Introduction of a de novo bioremediation ability, aryl reductive dechlorination, into anaerobic granular sludge by inoculation of sludge with Desulfomonile tiedjei.

Methanogenic upflow anaerobic granular-sludge blanket (UASB) reactors treat wastewaters at a high rate while simultaneously producing a useful product, methane; however, recalcitrant environmental pollutants may not be degraded. To impart 3-chlorobenzoate (3-CB)-dechlorinating ability to UASB reactors, we inoculated granular sludge in UASB reactors with either a pure culture of Desulfomonile tiedjei (a 3-CB-dechlorinating anaerobe) or a three-member consortium consisting of D. tiejei, a benzoate degrader, and an H2-utilizing methanogen. No degradation occurred in an uninoculated control reactor which was started with the same granular sludge, but inoculated reactors and granules from the inoculated UASB systems rapidly transformed 3-CB (54 mumol/day/g of granule biomass). After several months at a hydraulic retention time of 0.5 day, much shorter than the generation time of D. tiedjei, the reactors still dechlorinated 3-CB. This indicated that the bacteria were immobilized in the reactor granules, and by using an antibody probe for D. tiedjei, we demonstrated that this microorganism had colonized the sludge granules. These results represent the first addition of a pure culture or a defined microbial mixture to a viable waste treatment process to introduce a specific de novo degradative pathway into a granular-sludge consortium.     

Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor.

A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W. M. Wiegant and A. W. A. de Man, Biotechnol. Bioeng. 28:718-77, 1986) for production of granules dominated by Methanothrix spp. The reactor was inoculated with thermophilic digested sludge. No granules were observed during the first 7 months of start-up of the UASB reactor. However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material. After several years of stable operation, the effect of removing 60% of the granular sludge was investigated. Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1. These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically. The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration. The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities.     

An original procedure for physical simulation of upflow anaerobic sludge blanket reactors

A procedure for sludge blanket reactors physical simulation is presented. To simulate the liquid phase, a solution of 1.0 g/l of polyvinyl alcohol provided the desired rheological behavior. Biological granules presented in UASB reactors were simulated using granules of a water absorbent acrylic polymer capable to expand up to one hundred times in volume when moistened. The biochemical reactions that produces methane and carbon dioxide in anaerobic reactors were simulated reacting nitric acid and sodium bicarbonate. This procedure differs from gas injection since the gas is produced at the reactor core as it happens in the actual situation, thus the simulation is more realistic. Flow visualization and residence time distribution measurements were used to verify if the proposed procedure acceptably simulates UASB reactors treating wastewater. All tests were performed in a 10.5 l bench scale reactor and the results allow to recommend this method whenever flow visualization and reactor physical simulation are useful for reactor design and development.     

Stability studies of thermophilic anaerobic sludges under suboptimal feeding conditions and temperatures

The behaviour of thermophilic anaerobic sludge from the UASB process was investigated under suboptimal conditions. Stability studies, carried out during the normal operation of the reactors, showed that a short-term decrease in temperature had no influence on the behaviour of the process. Nevertheless, interruptions in feeding and a long-term decrease in temperatures can produce very drastic effects on the stability of the system.     

Layered structure of UASB granules gives microbial populations resistance to toxic chemicals

To investigate the effect of granular structure on resistance to toxic chemicals in UASB (Upflow Anaerobic Sludge Blanket) reactors, normal and broken granules were examined for their ability to degrade acetate with and without the addition of toluene or trichloroethylene as a toxic chemical. Without a toxic chemical, both normal and broken granules degraded the acetate at the same volumetric degradation rate (3.21 mM h–1). However, when 500 l l–1 of toluene or trichloroethylene was added, the acetate-degradation rate of the broken granules was about a third of the rate with normal granules. Therefore, the layered structure of the UASB granules seems to give microbial populations the ability to resist toxic chemicals.     

Distribution and change of microbial activity in combined UASB and AFB reactors for wastewater treatment

A thermophilic upflow anaerobic sludge blanket (UASB) reactor was combined with a mesophilic aerobic fluidized bed (AFB) reactor for treatment of a medium strength wastewater with 2,700?mg COD?l^?1. The COD removal efficiency reached 75% with a removal rate of 0.2 g COD?l^?1 h^?1 at an overall hydraulic retention time 14 hours. The distribution of microbial activity and its change with hydraulic retention time in the two reactors were investigated by measuring ATP concentration in the reactors and specific ATP content of the biomass. In the UASB reactor, the difference in specific ATP was significant between the sludge bed and blanket solution (0.02?mg ATP g VS^?1 versus 0.85?mg ATP g VS^?1) even though the ATP concentrations in these two zones were similar. A great pH gradient up to 4 was developed along the UASB reactor. Since a high ATP or biological activity in the blanket solution could only be maintained in a narrow pH range from 6.5 to 7.5, the sludge granules showed a high pH tolerance and buffering capacity up to pH 11. The suspended biomass in AFB reactor had a higher specific ATP than the biomass fixed in polyurethane carriers (1.6?mg ATP g VS^?1 versus 1.1?mg ATP g VS^?1), which implies a starvation status of the immobilized cells due to mass transfer limitation. The aerobes had to work under starvation conditions in this polishing reactor. The anaerobic biomass brought into AFB reactor contributed to an increase in suspended solids, but not the COD removal because of its fast deactivation under aerobic conditions. A second order kinetic model was proposed for ATP decline of the anaerobes. The results on distribution of microbial activity in the two reactors as well as its change with hydraulic retention time lead to further performance improvement of the combined anaerobic/aerobic reactor system.     

Granulation of biomass in thermophilic upflow anaerobic sludge blanket reactors treating acidified wastewaters

The development of granular sludge in thermophilic (55 degrees C) upflow anaerobic sludge blanket reactors was investigated. Acetate and a mixture of acetate and butyrate were used as substrates, serving as models for acidified waste-waters. Granular sludge with either Methanothrix or Methanosarcina as the predominant acetate utilizing methanogen was cultivated by allowing the loading rate to increase whenever the acetate concentration in the effluent dropped below 200 and 700 mg COD/L, respectively. The highest methane generation rates, up to 162 kg CH(4)-COD/m(3) day, or 2.53 mole CH(4)/L day, were achieved at hydraulic retention times down to 21 min, with granules consisting of Methanothrix. The formation of Methanothrix granules did not depend on the type of seed material, nor on the addition of inert support particles. The growth of granules proceeded rapidly with adapted seed material, even when the reactors were inoculated with low concentrations. With mesophilic seed materials growth of granules took much longer. Thermophilic Methanothrix granules strongly resemble mesophilic granules of the "filamentous" type. Some factors governing the thermophilic granulation process are discussed.     

High-rate treatment of terephthalate in anaerobic hybrid reactors.

The anaerobic degradation of terephthalate as sole substrate was studied in three anaerobic upflow reactors. Initially, the reactors were operated as upflow anaerobic sludge bed (UASB) reactors and seeded with suspended methanogenic biomass obtained from a full-scale down-flow fixed film reactor, treating wastewater generated during production of purified terephthalic acid. The reactors were operated at 30, 37, and 55 degrees C. The terephthalate removal capacities remained low in all three reactors (<4 mmolxL-1xday-1, or 1 g of chemical oxygen demand (COD)xL-1xday-1) due to limitations in biomass retention. Batch experiments with biomass from the UASB reactors revealed that, within the mesophilic temperature range, optimal terephthalate degradation is obtained at 37 degrees C. No thermophilic terephthalate-degrading culture could be obtained in either continuous or batch cultures. To enhance biomass retention, the reactors were modified to anaerobic hybrid reactors by introduction of two types of reticulated polyurethane (PUR) foam particles. The hybrid reactors were operated at 37 degrees C and seeded with a mixture of biomass from the UASB reactors operated at 30 and 37 degrees C. After a lag period of approximately 80 days, the terephthalate conversion capacity of the hybrid reactors increased exponentially at a specific rate of approximately 0.06 day-1, and high removal rates were obtained (40-70 mmolxL-1xday-1, or 10-17 g of CODxL-1xday-1) at hydraulic retention times between 5 and 8 h. These high removal capacities could be attributed to enhanced biomass retention by the development of biofilms on the PUR carrier material as well as the formation of granular biomass. Biomass balances over the hybrid reactors suggested that either bacterial decay or selective wash-out of the terephthalate fermenting biomass played an important role in the capacity limitations of the systems. The presented results suggest that terephthalate can be degraded at high volumetric rates if sufficiently long sludge ages can be maintained, and the reactor pH and temperature are close to their optima.     

Nutrient requirement for UASB process: a review

In literature, numerous studies regarding nutrients dosing are available in UASB reactors. Various nutrients and tracemetals used in UASB studies include nitrogen, phosphorous, yeast extract, magnesium, potassium, calcium, sulphur, iron, aluminium, zinc, nickel, cobalt, molybdenum, manganese, copper, boron, selenium, citrate, resazurine, EDTA, and hydrochloric acid. Often, there is a wide variation in the composition and quantification of nutrients and tracemetals, used to supplement the influent feed in UASB reactors. In this paper, nutrients and tracemetals formulations used in different studies on UASB reactors are compiled and critically analysed. In order to have uniformity, concentrations of nutrients and tracemetals have been converted into equivalent terms as g nutrients or tracemetals/g of waste COD. The ranges for this ratio for all the constituents of nutrients and tracemetals are given. Besides, the ranges associated with minimum start-up periods are also obtained. It is hoped that the information presented in this study would facilitate the nutrients dosing in UASB reactors.     

Microbial community changes in methanogenic granules during the transition from mesophilic to thermophilic conditions

Upflow anaerobic sludge blanket (UASB) reactor is one of the most applied technologies for various high-strength wastewater treatments. The present study analysed the microbial community changes in UASB granules during the transition from mesophilic to thermophilic conditions. Dynamicity of microbial community in granules was analysed using high-throughput sequencing of 16S ribosomal RNA gene amplicons, and the results showed that the temperature strictly determines the diversity of the microbial consortium. It was demonstrated that most of the microbes which were present in the initial mesophilic community were not found in the granules after the transition to thermophilic conditions. More specifically, only members from family Anaerolinaceae managed to tolerate the temperature change and contributed in maintaining the physical integrity of granular structure. On the contrary, new hydrolytic and fermentative bacteria were quickly replacing the old members in the community. A direct result from this abrupt change in the microbial diversity was the accumulation of volatile fatty acids and the concomitant pH drop in the reactor inhibiting the overall anaerobic digestion process. Nevertheless, by maintaining deliberately the pH levels at values higher than 6.5, a methanogen belonging to Methanoculleus genus emerged in the community enhancing the methane production.

     

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Reactivation of effluent granular sludge from a high-rate Anammox reactor after storage

In this study, effluent sludge from a high-rate Anammox reactor was used to re-start new Anammox reactors for the reactivation of Anammox granular sludge. Different start-up strategies were evaluated in six upflow anaerobic sludge blanket (UASB) reactors (R₁–R₆) for their effect on nitrogen removal performance. Maximal nitrogen removal rates (NRRs) greater than 20 kg N/m³/day were obtained in reactors R₃–R₅, which were seeded with mixed Anammox sludge previously stored for approximately 6 months and 1 month. A modified Boltzmann model describing the evolution of the NRR fit the experimental data well. An amount of sludge added to the UASB reactor or decreasing the loading rate proved effective in relieving the substrate inhibition and increasing the NRR. The modified Stover–Kincannon model fit the nitrogen removal data in the Anammox reactors well, and the simulation results showed that the Anammox process has great nitrogen removal potential. The observed inhibition in the Anammox reactors may have been caused by high levels of free ammonia. The sludge used to seed the reactors did not settle well; sludge flotation was observed even after the reactors were operated for a long time at a floating upward velocity (F_s) of greater than 100 m/h. The settling sludge, however, exhibited good settling properties. Scanning electron microscopy showed that the Anammox granules consisted mainly of spherical and elliptical bacteria with abundant filaments on their surface. Hollows in the granules were also present, which may have contributed to sludge floatation.