Long-term, high-rate anaerobic biological treatment of whey wastewaters at psychrophilic temperatures

Two laboratory-scale anaerobic hybrid reactors, R1 and R2, treated low- (1 kg COD m-3) and high-strength (10 kg COD m-3) whey-based wastewaters, respectively, in a 500-day trial. The chemical oxygen demand (COD) removal efficiencies of R1 averaged 70-80%, at organic loading rates of 0.5-1.3 kg COD m-3 day-1, between 20 and 12 degrees C. The COD removal efficiencies of R2 exceeded 90%, at organic loading rates up to 13.3 kg COD m-3 day-1, between 20 and 14 degrees C. Lowering the operating temperature of R2 to 12 degrees C resulted in a decrease in COD removal efficiency, to between 50% and 60%, and a disintegration of granular sludge. The decline in performance, and granule disintegration, was reversed by decreasing the organic loading rate of R2 to 6.6 kg m-3 day-1. Specific methanogenic activity profiles revealed mesophilic (37 degrees C) temperature optima for biomass in both reactors, even after 500 days of psychrophilic operation, although the development of psychrotolerance in the biomass was noted.     

Temporal microbial diversity changes in solvent-degrading anaerobic granular sludge from low-temperature (15 degrees C) wastewater treatment bioreactors

Anaerobic sludge granules were obtained from laboratory-scale anaerobic bioreactors used to treat pharmaceutical-like (methanol-, acetone- and propanol-contaminated) wastewater under low-temperature conditions (15 degrees C). The microbial diversity and diversity changes of the sludge samples were ascertained by applying 16S rRNA gene cloning and terminal restriction fragment length polymorphism (TRFLP) analyses, respectively, and using sludge samples from the inoculum, throughout and at the conclusion of the bioreactor trial. Data from genetic fingerprinting correlated well with those from physiological activity assays of the reactor biomass. Specifically, for example, TRFLP profiles indicated the dominance of hydrogenotrophic methanogens within the archaeal community, thus supporting the findings of specific methanogenic activity measurements. TRFLP data supported the hypothesis that the deviation between the replicated reactors, in terms of treatment efficiency, was associated with succession within the microbial communities present, and indicated that community development was linked to both operating temperature and wastewater composition. Fluorescence in situ hybridization (FISH) was also applied, to quantitatively assess the abundance of selected microbial groups, and revealed the underestimation of the abundance Methanosarcina by gene cloning analysis and demonstrated the spatial arrangement of these organisms within the architecture of the low-temperature solvent-degrading anaerobic biofilms.     

Effects of an electric field and zero valent iron on anaerobic treatment of azo dye wastewater and microbial community structures

A zero valent iron (ZVI) bed with a pair of electrodes was packed in an anaerobic reactor aiming at enhancing treatment of azo dye wastewater. The experiments were carried out in three reactors operated in parallel: an electric field enhanced ZVI-anaerobic reactor (R1), a ZVI-anaerobic reactor (R2) and a common anaerobic reactor (R3). R1 presented the highest performance in removal of COD and color. Raising voltage in R1 further improved its performance. Scanning electron microscopy images displayed that the structure of granular sludge from R1 was intact after being fed with the high dye concentration, while that of R3 was broken. Fluorescence in situ hybridization analysis indicated that the abundance of methanogens in R1 was significantly greater than that in the other two reactors. Denaturing gradient gel electrophoresis showed that the coupling of electric field and ZVI increased the diversity of microbial community and especially enhanced bacterial strains responsible for decolorization.     

Effect of effluent recycling and shock loading on the biodegradation of complex phenolic mixture in hybrid UASB reactors

This study describes the feasibility of anaerobic treatment of synthetic coal wastewater using four identical 13.5L (effective volume) bench scale hybrid up flow anaerobic sludge blanket (HUASB) reactors (R1, R2, R3 and R4) under mesophilic (27+/-5 degrees C) conditions. Synthetic coal wastewater with an average chemical oxygen demand (COD) of 2240 mg/L and phenolics concentration of 752 mg/L was used as substrate. Effluent recirculation was employed at four different effluent to feed recirculation ratios (R/F) of 0.5, 1.0, 1.5 and 2.0 for 100 days to study the effect of recirculation on the performance of the reactors. Phenolics and COD removal was found to improve with increase in effluent recirculation. An effluent to feed recycle ratio of 1.0 resulted in maximum removal of phenolics and COD. Phenolics and COD removal improved from 88% and 92% to 95% each, respectively. The concentration of volatile fatty acids in the effluent was lower than the influent when effluent to feed recirculation was employed. Effect of shock loading on the reactors revealed that phenolics shock load up to 2.5 times increase in the normal input phenolics concentration in the form of continuous shock load for 4days did not affect the reactors performance irreversibly.     

Novel application of oxygen-transferring membranes to improve anaerobic wastewater treatment

Anaerobic biological wastewater treatment has numerous advantages over conventional aerobic processes; anaerobic biotechnologies, however, still have a reputation for low-quality effluents and operational instabilities. In this study, anaerobic bioreactors were augmented with an oxygen-transferring membrane to improve treatment performance. Two anaerobic bioreactors were fed a synthetic high-strength wastewater (chemical oxygen demand, or COD, of 11,000 mg l(-1)) and concurrently operated until biomass concentrations and effluent quality stabilized. Membrane aeration was then initiated in one of these bioreactors, leading to substantially improved COD removal efficiency (> 95%) compared to the unaerated control bioreactor (approximately 65%). The membrane-augmented anaerobic bioreactor required substantially less base addition to maintain circumneutral pH and exhibited 75% lower volatile fatty acid concentrations compared to the unaerated control bioreactor. The membrane-aerated bioreactor, however, failed to improve nitrogenous removal efficiency and produced 80% less biogas than the control bioreactor. A third membrane-augmented anaerobic bioreactor was operated to investigate the impact of start-up procedure on nitrogenous pollutant removal. In this bioreactor, excellent COD (>90%) and nitrogenous (>95%) pollutant removal efficiencies were observed at an intermediate COD concentration (5,500 mg l(-1)). Once the organic content of the influent wastewater was increased to full strength (COD = 11,000 mg l(-1)), however, nitrogenous pollutant removal stopped. This research demonstrates that partial aeration of anaerobic bioreactors using oxygen-transferring membranes is a novel approach to improve treatment performance. Additional research, however, is needed to optimize membrane surface area versus the organic loading rate to achieve the desired effluent quality.     

Anaerobic granule development for removal of pentachlorophenol in an upflow anaerobic sludge blanket (UASB) reactor

The granulation process using synthetic wastewater containing pentachlorophenol (PCP) in four 1.1 l laboratory scale upflow anaerobic sludge blanket (UASB) reactors was studied, and the anaerobic biotransformation of PCP during the granulation process investigated. After 110 days granular sludge was developed and up to 160 and 180 mg/l of PCP was added into the reactors R1 and R2, respectively, when they were inoculated with acclimated anaerobic sludge from an anaerobic digester of a citric acid plant. The inoculum was predominately composed of bacilli and filamentous bacteria. Granulation did not occur in reactors R3 and R4 which were inoculated with acclimated anaerobic sludge from aerobic sludge of the municipal sewage treatment plant which consisted mainly of cocci. Despite similar bacilli in the granule, the filamentous bacteria from reactor R1 were thicker than those of reactor R2. The granular sludge had a maximum diameter of 2.5 and 2.2 mm, and SMA of 1.44 and 1.32 gCOD/gTVS per day for reactors R1 and R2, respectively. Over 98% chemical oxygen demand (COD) removal rate and 99% of PCP removal rate were achieved when reactors R1 and R2 were operated at PCP and COD loading rates of 150 and 7.5 g/l per day, respectively. H₂-producing acetogens were the dominant anaerobes in the granular sludge.     

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.     

Inhibition of mesophilic solid-substrate anaerobic digestion by ammonia nitrogen

This work focused on determining the effects of ammonia-nitrogen supplementation on the mesophilic solid-substrate anaerobic digestion of municipal wastes and waste activated sludge (biosolids). Bench-scale, semi-continuous, mesophilic reactors were operated with a 21-day mass-retention time and dosed with NH₄Cl, such that the corresponding chemical O₂ demand (COD)/N ratios in their feeds were 90, 80, 65 and 50 (reactors R1 or control, R2, R3 and R4 respectively). Reactor performance was evaluated in terms of the efficiency of volatile solid removal (efficiency for short), biogas productivity, methane content in the biogas, pH and volatile organic acid contents, among other monitoring and analytical parameters. The feedstock was a mixture of urban wastes with biosolids. It was found that the process performance deteriorated at increasing dosages of ammonia N, the process practically ceasing at COD/N = 50 (R4). Inhibition was characterized by efficiency and biogas productivity decreases and a more sudden drop of methane content in biogas and pH. A significant rise of propionic, butyric and valeric acid was found in reactors receiving the highest doses of ammonia N (R3 and R4). This suggested that inhibition of the syntrophic bacteria present in the anaerobic consortia also occurred. Luong and Pearson inhibition models were fitted to the data. Both models represented very well the acute effects of N supplementation on solid-substrate anaerobic digestion. However, the Luong model could also represent the process ceasing at a critical ammonia N concentration of 2800 mg/kg mixed solids. Received: 12 April 1996 / Received revision: 23 July 1996 / Accepted: 5 August 1996     

Granule development and performance in sucrose fed anaerobic baffled reactors

Two 90 L anaerobic baffled reactors were used to study the granulation of sludge and the effect of the organic loading rate and NaHCO3/COD ratios on reactor performance. Furthermore, it was determined whether an anaerobic baffled reactor would promote phase separation and if additive of bentonite or granular active carbon was capable of enhancing granule formation. In order to minimize feed variations, and have a totally biodegradable substrate, a synthetic sucrose substrate was used. Granulation was achieved in both reactors within 75 days. However, the granules from the granular active carbon amended reactor appeared earlier and were larger and more compact. The reactors were maintained at a hydraulic retention time of 20 h during performance study stage. The results showed that when organic loading rate were changed from 2.15 to 6.29 kg COD m(-3)day(-1), chemical oxygen demand (COD) removal was not decreased (91-93%), but a slight increase in effluent COD was observed. It was found that the COD removals were generally good (87-92%) and had not obviously change with the decreasing NaHCO3/COD ratios. From the bacterial distribution and the concentration of volatile fatty acids in four compartments, it was concluded that a separation of phases occurred within the anaerobic baffled reactors.     

三种接种物启动Anammox-EGSB反应器的性能

为了优选接种物和加速Anammox反应器启动,分别以厌氧产甲烷污泥 (Anaerobic methanogenic sludge,AMS)、新鲜厌氧氨氧化污泥 (Fresh Anammox sludge,FAS) 和储藏厌氧氨氧化污泥 (Stored Anammox sludge,SAS) 作为接种物,研究了厌氧氨氧化膨胀颗粒污泥床 (Anammox-EGSB) 反应器 (R1、R2和R3) 的启动性能。结果表明：3种接种物均能成功启动Anammox-EGSB反应器,启动性能的优劣次序为：R2 (接种物为     

Biological treatment of wastewater containing an azo dye using mixed culture in alternating anaerobic/aerobic sequencing batch reactors

The performance of one stage anaerobic/aerobic processes for the biological treatment of synthetic wastewaters containing Acid Red 18 was studied. In addition, a method for evaluating dye mineralization using lumped parameters was investigated. The selected initial dye concentrations were 0, 35, 70, 140, and 280 mg/L, in reactors R1 ～ R5, respectively. This study showed that average COD removal was not lower than 85% while the remaining COD originated from Acid Red 18 and its degradation products. The majority of the dye removal occurred in the anaerobic phases and the aerobic phase contributions were insignificant. The kinetics data of dye removal showed that the increase in initial dye concentration (35 ～ 280 mg/L) caused a decrease in first-order kinetic rate constants (0.0593 ～ 0.0384/h). The overall mineralization of AR 18 dye was at least 44, 35, 13, and 0% in R2 ～ R5, respectively. Increase in initial dye concentrations had no significant effect on sludge characteristics.     

Biodegradation of olive-mill pomace mixed with organic fraction of municipal solid waste

This study investigated the effects of organic fraction of municipal solid waste (OFMSW) addition on the anaerobic treatment of the olive-mill pomace. Biodegradability of olive-mill pomace mixed with OFMSW was examined in anaerobic bioreactors. Only OFMSW was loaded in the first (control) bioreactor, while run 1 and run 2 bioreactors included different ratio of OFMSW and olive-mill pomace. COD, BOD₅, NH₄–N, pH, VFA, CH₄ quantity and percentage in anaerobic bioreactors were regularly monitored. In addition, inert COD and anaerobic toxicity assay (ATA) were measured in leachate samples. The results of the study showed that 70% of OFMSW addition to olive-mill pomace has an advantage in terms of pollution parameters and methane generation. Since olive-mill pomace is not easy biodegradable, addition of high proportion of OFMSW promotes biodegradability of olive-mill pomace. Decreasing in BOD₅/COD ratios in the run 1 and run 2 reactors carried out as 62 and 52%, respectively.     

Treatment of slaughterhouse wastewater in a UASB reactor and an anaerobic filter

A study was performed to assess the feasibility of anaerobic treatment of slaughterhouse wastewaters in a UASB (Upflow Anaerobic Sludge Blanket) reactor and in an AF (Anaerobic Filter). Among the different streams generated, the slaughter line showed the highest organic content with an average COD of 8000 mg/l, of which 70% was proteins. The suspended solids content represented between 15 and 30% of the COD. Both reactors had a working volume of 21. They were operated at 37°C. The UASB reactor was run at OLR (Organic Loading Rates) of 1–6.5 kg COD/m³/day. The COD removal was 90% for OLR up to 5 kg COD/m³/day and 60% for an OLR of 6.5 kg COD/m³/day. For similar organic loading rates, the AF showed lower removal efficiencies and lower percentages of methanization. At higher OLR sludge, flotation occurred and consequently the active biomass was washed out from the filter. The results indicated that anaerobic treatment systems are applicable to slaughterhouse wastewaters and that the UASB reactor shows a better performance, giving higher COD removal efficiencies than the AF.     

Molecular weight distribution of Pinus radiata kraft mill wastewater treated by anaerobic digestion

Kraft mill is responsible for massive discharge of highly polluted effluents. The main characteristics of this effluent are high toxicity and low biodegradability due to tannin, lignin and chlorophenol compounds. The composition may vary dramatically depending, for instance, on the utilised feedstock and process. The purpose of this work was to investigate the molecular weight distribution of Pinus radiata kraft pulping wastewater treated by anaerobic digestion by using two types of anaerobic reactors: fixed bed and sludge blanket. Anaerobic sludge blanket (UASB) and anaerobic filter (AF) were operated. In both reactors, the total alkalinity ranged between 1.0 and 1.5 g CaCO3/l, while the organic load rate (OLR) was increasing during operation from 1.2 to 3.3 gCOD/l d. COD and total phenolic compounds (UV215) removal ranged between 30-50% and 13-20%, respectively, while the BOD5 removal ranged 60-90%. However only a partial biodegradation (10-43%) of tannin and lignin was observed. Results from ultrafiltration analyses indicated that the fraction with a molecular weight (MW) < 1000, COD and colour decreased after anaerobic treatment, but the total phenolic compounds increased. In the 1000 < MW < 10,000 fraction, there was no change in COD, UV215 and colour. In the > 10,000 MW fraction, colour and COD fraction increased by 14% and 5%, respectively, after anaerobic treatment. It can be concluded from this study, that treatment with UASB or AF reactors is not enough, under the conditions tested, for a large COD removal from Pinus radiata wastewater.     

Effects of alternative carbon sources on biological transformation of nitrophenols

The removal of nitrophenols under denitrifying conditions was studied in bench-scale upflow anaerobic sludge blanket (UASB) reactors (R1, R2, R3 and R4) using three different carbon sources. Initially acetate was used as carbon source (substrate) in all the four reactors followed by glucose and methanol. Reactor R1 was kept as control and R2, R3, R4 were fed with 30 mg/l concentration of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. Throughout the study the hydraulic retention time (HRT) and COD/NO₃ ^-–N ratio were kept as 24 h and 10, respectively. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolites of 2-NP, 4-NP and 2,4-DNP degradation, respectively. Methanol was found to be a better carbon source for 4-NP and 2,4-DNP degradation as compared to acetate and glucose, while 2-NP degradation was not influenced much by the change of substrate. Nitrate nitrogen removal was always more than 99%. COD removal efficiency of the nitrophenol fed reactors varied from 85.7% to 97.7%. The oxidation-reduction potential (ORP) inside the reactors dropped, up to –300 mv, with glucose as carbon source. As the reactors were switched over to methanol, ORP increased to –190 mv. The granular sludge developed inside the reactors was light brown in colour when acetate and glucose were used as substrate, which turned dark brown to black at the end of methanol run. Biomass yield in terms of volatile suspended solids was observed as 0.15, 0.089 and 0.14 g per gram of COD removal for acetate, glucose and methanol, respectively.     

Granulation in anaerobic sludge bed reactors treating food industry wastes

Three parallel laboratory reactors, R1, R2 and R3, received food industry wastewater: R1 unadulterated; R2 supplemented with calcium and phosphate; R3 supplemented with ferric chloride and traces of nickel and cobalt. Reactors were packed with active granular sludge from a large scale pilot reactor treating the same wastewater. Addition of calcium and phosphate was found to be detrimental to the granule formation at naturally established reactor pH = 6·9–7·4 in R2 while iron promoted granulation in R3. Conditions of upflow velocities of 1·5–6 m h⁻¹, rapid increase of loads up to 15 kg COD m⁻³ day⁻¹ and ratios of recycle to raw waste feed of 20:1–80:1 were imposed on all reactors. The granules in R1 and R2 disintegrated, from 70–100 g liter⁻¹ VSS to a flocculant sludge at 1·5–3 g liter⁻¹. In spite of such severe washout, reactors R1 and R2 were able to maintain a steady COD removal of over 90% at a load of 10kg m⁻³ day⁻¹. R3 retained a VSS concentration around 100 g liter⁻¹ and maintained COD removal at over 95%. R3 exhibited a more stable performance and was less vulnerable to the shock treatment to which all reactors were subjected.     

Systematic analysis of biochemical performance and the microbial community of an activated sludge process using ozone-treated sludge for sludge reduction

Two lab-scale bioreactors (reactors 1 and 2) were employed to examine the changes in biological performance and the microbial community of an activated sludge process fed with ozonated sludge for sludge reduction. During the 122 d operation, the microbial activities and community in the two reactors were evaluated. The results indicated that, when compared with the conventional reactor (reactor 1), the reactor that was fed with the ozonated sludge (reactor 2) showed good removal of COD, TN and cell debris, without formation of any excess sludge. In addition, the protease activity and intracellular ATP concentration of reactor 2 were increased when compared to reactor 1, indicating that reactor 2 had a better ability to digest proteins and cell debris. DGGE analysis revealed that the bacterial communities in the two reactors were different, and that the dissimilarity of the bacterial population was nearly 40%. Reactor 2 also contained more protozoa and metazoa, which could graze on the ozone-treated sludge debris directly.     

固定载体卧式厌氧反应器处理糖蜜废水的快速启动

为高效处理高浓度有机废水而设计了固定载体卧式厌氧反应器R1和R2, 它是厌氧折流板反应器(ABR)的改进, 以活性炭纤维作为生物膜载体固定并充当反应器的折流板, 在实验室规模上对R1和R2处理糖蜜废水进行快速启动运行。HRT和ORL是影响R1和R2稳定高效运行及启动的2个重要工艺参数。实验证明: HRT为2 d时, 反应器运行最佳。在第30天时, R1的COD去除率达到84.88%, R2达到81.72%。随着进水ORL由1.25 kg/(m3·d)提升到10 kg/(m3·d), 沼气容积产气率由0.35 L/(L·d)逐渐增加到4.98 L/(L·d)。进水pH值为3.9?4.5之间, 整个启动运行过程中, 未调节pH值, R1和R2的出水pH值均在6.7?7.6之间, 2个反应器均有较强的抗酸能力, R1的pH波动更为平缓。在整个实验过程中, 污泥流失量小, 没有发生堵塞现象, 在处理酸性高浓度有机废水时, 2个反应器均表现出较强的抗负荷冲击能力。     

Enhanced aerobic granulation and nitrogen removal by the addition of zeolite powder in a sequencing batch reactor

The aim of this study was to evaluate the impact of zeolite powders on feasibility of rapid aerobic granulation in the column-type sequencing batch reactors. After 90 days' operation, aerobic granular sludge was formed in both reactors by altering influent chemical oxygen demand/nitrogen (COD/N) ratios. R1 with zeolite powders had better removal capabilities of COD and total nitrogen than R2, which was without zeolite powders. Mixed liquor volatile suspended solid concentrations of the two reactors were 7.36 and 5.45 g/L, while sludge volume index (SVI₃₀) values were 34.9 and 47.9 mg/L, respectively. The mean diameters of aerobic granular sludge in the above two reactors were 2.5 and 1.5 mm, respectively. Both reactors achieved the largest simultaneous nitrification and denitrification (SND) efficiency at an influent COD/N ratio of 8; however, R1 exhibited more excellent SND efficiency than R2. The obtained results could provide a novel technique for rapid aerobic granulation and N removal simultaneously, especially when treating nitrogen-rich industrial wastewater.     

Biotransformation of nitrophenols in upflow anaerobic sludge blanket reactors

Four identical bench-scale upflow anaerobic sludge blanket (UASB) reactors, R1, R2, R3 and R4, were used to assess nitrophenols degradation at four different hydraulic retention times (HRT). Reactor R1 was used as control, whereas R2, R3, and R4 were fed with 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. The concentration of each nitrophenol was gradually varied from 2 to 30 mg/l during acclimation. After acclimation reactors were operated under steady-state conditions at four different HRTs – 30, 24, 18, and 12 h, to study its effect on the removal of nitrophenols. Overall removal of 2-NP and 4-NP was always more than 99% but 2,4-DNP removal decreased from 96% to 89.7% as HRT was lowered from 30 to 12 h. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found to be the major intermediates during the degradation of 2-NP, 4-NP and 2,4-DNP, respectively. Out of the total input of nitrophenolic concentration (30 mg/l), on molar basis, about 41.2–48.4% of 2-NP, 59.4–68% of 4-NP, 30–26.6% of 2,4-DNP was recovered in the form of their respective amino derivatives at 30–12 h HRT. COD removal was 98–89%, 97–56%, 97–52%, and 94–46% at 30–12 h HRT for R1, R2, R3 and R4, respectively. Average cell growth was observed to be 0.15 g volatile suspended solid (VSS) per g COD consumed. Methanogenic inhibition was observed at lower HRTs (18 and 12 h), however denitrification was always more than 99% with non-detectable level of nitrite. The granules developed inside the reactors were black in color and their average size varied between 1.9 and 2.1 mm.