Denitrification on poly-beta-hydroxybutyrate in microbial granular sludge sequencing batch reactor

Microbial granules were successfully cultivated in an alternating aerobic-anaerobic sequencing batch reactor (SBR) for removing organic carbon and nitrogen. It was found that almost all input ammonium was converted to nitrite and nitrate in the aerobic phase, while the efficiency of denitrification was highly related to the availability of external carbon source in the anaerobic phase. Complete denitrification was achieved with sufficient supply of external carbon, while only partial denitrification was observed with no addition of external carbon. Results showed that in the absence of external carbon source, pre-accumulated poly-beta-hydroxybutyric acid (PHB) in microbial granules could be utilized for cell maintenance and denitrification. With supply of external carbon but no addition of nitrate, PHB accumulation accounted for the main mechanism of the organic removal. Under balanced growth conditions (with organic carbon and nitrogen supply), external organic carbon was consumed simultaneously for denitrification, PHB storage as well as for cell functions. It was revealed that the potential role of PHB for denitrification by microbial granules was very limited, i.e. less than 28 mg nitrate-nitrogen l(-1) was found to be denitrified with internally accumulated PHB. This study for the first time shows the limiting capacity of PHB as reducing power for denitrification by microbial granules.     

Effects of oxygen on biodegradation of benzoate and 3-chlorobenzoate in a denitrifying chemostat

A mixed microbial culture degraded a mixture of benzoate (863 mg/L), 3-chlorobenzoate (3-CB) (69.7 mg/L), and pyruvate (244 mg/L) under denitrifying conditions in a chemostat. Biodegradation under denitrifying conditions was stable, complete (effluent concentrations below detection limits), and proceeded without the production of toxic intermediates like chlorocatechols. The addition of oxygen at mass input rates of 6.2%, 15.5%, and 43.9% of the mass input rate of chemical oxygen demand (COD) (337 mg COD/h) did not induce the synthesis of aerobic biodegradation pathways and thus did not disrupt biodegradation. Rather, the oxygen was used as a terminal electron acceptor, displacing a stoichiometric amount of nitrate, leading to microaerobic conditions (dissolved oxygen concentration <0.050 mg/L) in which oxygen utilization and denitrification occurred simultaneously. The reduction of nitrate occurred fully to N₂ gas with no accumulation of nitrite, nitrous oxide, or nitric oxide, although the ability of the culture to transfer electrons to the nitrogen oxides decreased as the oxygen input was increased. The anoxic benzoate uptake capability was unaffected by the increase in oxygen addition, but the anoxic 3-CB uptake capability increased, as did the level of benzoyl-CoA reductase in the cells.     

Optimized aeration strategies for nitrogen and phosphorus removal with aerobic granular sludge

Biological wastewater treatment by aerobic granular sludge biofilms offers the possibility to combine carbon (COD), nitrogen (N) and phosphorus (P) removal in a single reactor. Since denitrification can be affected by suboptimal dissolved oxygen concentrations (DO) and limited availability of COD, different aeration strategies and COD loads were tested to improve N- and P-removal in granular sludge systems. Aeration strategies promoting alternating nitrification and denitrification (AND) were studied to improve reactor efficiencies in comparison with more classical simultaneous nitrification–denitrification (SND) strategies. With nutrient loading rates of 1.6 gCOD L⁻¹ d⁻¹, 0.2 gN L⁻¹ d⁻¹, and 0.08 gP L⁻¹ d⁻¹, and SND aeration strategies, N-removal was limited to 62.3 ± 3.4%. Higher COD loads markedly improved N-removal showing that denitrification was limited by COD. AND strategies were more efficient than SND strategies. Alternating high and low DO phases during the aeration phase increased N-removal to 71.2 ± 5.6% with a COD loading rate of 1.6 gCOD L⁻¹ d⁻¹. Periods of low DO were presumably favorable to denitrifying P-removal saving COD necessary for heterotrophic N-removal. Intermittent aeration with anoxic periods without mixing between the aeration pulses was even more favorable to N-removal, resulting in 78.3 ± 2.9% N-removal with the lowest COD loading rate tested. P-removal was under all tested conditions between 88 and 98%, and was negatively correlated with the concentration of nitrite and nitrate in the effluent (r = −0.74, p < 0.01). With low COD loading rates, important emissions of undesired N₂O gas were observed and a total of 7–9% of N left the reactor as N₂O. However, N₂O emissions significantly decreased with higher COD loads under AND conditions.     

Nitrogen and phosphorus removal from an abattoir wastewater in a SBR with aerobic granular sludge

The formation and performance of granular sludge was studied in an 8 l sequencing batch reactor (SBR) treating an abattoir (slaughterhouse) wastewater. Influent concentrations averaged 1520 mg l⁻¹ volatile suspended solids (VSS), 7685 mg l⁻¹ Chemical oxygen demand (COD), 1057 mg l⁻¹ total kjeldahl nitrogen (TKN), 217 mg l⁻¹ total P. The COD loading was 2.6 kg m⁻³ d⁻¹. The SBR was seeded with flocculating sludge from a SBR with an 1 h settle time, but granules developed within 4 days by reducing the settle time to 2 min. The SBR cycle also had 120 min mixed (anaerobic) fill, 220 min aerated react, and 18 min draw/idle. The granules had a mean diameter of 1.7 mm, a specific gravity of 1.035, a density of 62 g VSS l⁻¹, a zone settling velocity (ZSV) of 51 m h⁻¹, and a sludge volume index (SVI) of 22 ml g⁻¹. Without optimizing process conditions, removal of COD and P were over 98%, and removal of N and VSS were over 97%. Nitrification and denitrification occurred simultaneously during react. The results indicate that conventional SBRs treating wastewaters with flocculating sludge can be converted to granular SBRs by reducing the settle time.     

Cost-efficient operation of a denitrifying activated sludge process

In this paper, the choice of optimal set-points and cost minimizing control strategies for the denitrification process in an activated sludge process are treated. In order to compare different criterion functions, simulations utilizing the COST/IWA simulation benchmark are considered. By means of operational maps the results are visualized. It is found that it is easy to distinguish set-point areas where the process can be said to be efficiently controlled in an economic sense. The characteristics of these set-point areas depend on the chosen effluent nitrate set-point as well as the distribution of the different operating costs. It is also discussed how efficient control strategies may be accomplished.     

Accelerated start-up and enhanced granulation in upflow anaerobic sludge blanket reactors

In the present study, the effects of a cationic polymer on reactor start-up and granule development were evaluated. A control reactor R1 was operated without adding polymer, while the other five reactors designated R2, R3, R4, R5 and R6 were operated with different polymer concentrations of 20, 40, 80, 160 and 320 mg/L, respectively. Experimental results demonstrated that adding the polymer at a concentration of 80 mg/L markedly accelerated the start-up time. The time required to reach stable treatment at an organic loading rate (OLR) of 4 g COD/L.d was reduced by approximately 50% in R4 as compared with the control reactor. The same reactor with 80 mg/L polymer was able to achieve an OLR of 12 g COD/L.d after 59 days of operation, while R1, R2, R3, R5 and R6 achieved the same loading rate at much longer period of 104, 80, 69, 63 and 69 days, respectively. Comparing with the control reactor, the start-up time of R4 was shortened markedly by about 43% at this OLR, while other reactors also recorded varying degree of shortening. Monitoring on granule development showed that the granule formation was accelerated by 30% from the use of the appropriate dosage of polymer. Subsequent granules characterization indicated that the granules developed in R4 with 80 mg/L polymer exhibited the best settleability, strength and methanogenic activity at all OLRs. The organic loading capacities of reactors were also increased by the polymer addition. The maximum organic loading of the control reactor was 24 g COD/L.d, while the polymer-assisted reactor added with 80 mg/L polymer attained a markedly increased organic loading of 40 g COD/L.d. The laboratory results obtained demonstrated that adding the cationic polymer could result in shortening of start-up time and enhancement of granulation, which in turn lead to improvement in organics removal efficiency and loading capacity of the UASB system.     

Simultaneous nitrogen and phosphate removal in aerobic granular sludge reactors operated at different temperatures

The main biological conversions taking place in two lab-scale aerobic granular sludge sequencing batch reactors were evaluated. Reactors were operated at different temperatures (20 and 30 °C) and accomplished simultaneous COD, nitrogen and phosphate removal. Nitrogen and phosphate conversions were linked to the microbial community structure as assessed by fluorescent in situ hybridization (FISH) analysis. Anoxic tests were performed to evaluate the contribution of anoxic phosphate uptake to the overall phosphate removal and to clarify the denitrification pathway. Complete nitrification/denitrification and phosphate removal were achieved in both systems. A considerable fraction of the phosphate removal was coupled to denitrification (denitrifying dephosphatation). From the results obtained in anoxic batch experiments dosing either nitrite or nitrate, denitrification was proposed to proceed mainly via the nitrate pathway. Denitrifying glycogen-accumulating organisms (DGAOs) were observed to be the main organisms responsible for the reduction of nitrate to nitrite. A significant fraction of the nitrite was further reduced to nitrogen gas while being used as electron acceptor by denitrifying polyphosphate-accumulating organisms (PAO clade II) for anoxic phosphate uptake.     

Coupled BAS and anoxic USB system to remove urea and formaldehyde from wastewater

Wastewater containing formaldehyde and urea was treated using a coupled system consisting of a biofilm airlift suspension (BAS) reactor and an anoxic upflow sludge blanket (USB) reactor. The anoxic USB reactor was used to carry out denitrification and urea hydrolysis, while the BAS reactor was used to carry out nitrification. In a first step, individual experiments were carried out to investigate the effects of both compounds on the nitrifying and denitrifying biomass. The BAS reactor was fed with a synthetic medium containing 500 mg N-NH4(+)l(-1) and 100mg N-urea l(-1), that were added continuously to this medium. Neither urea hydrolysis nor inhibition of nitrification was observed. Nitrification efficiency decreased when formaldehyde was fed during shocks at concentrations of 40, 80 and 120 mg C-formaldehyde l(-1). The anoxic USB reactor was fed with a synthetic medium containing nitrate, formaldehyde and urea. Concentrations of formaldehyde in the reactor of 100-120 mg C-formaldehyde l(-1) caused a decrease in the denitrification and urea hydrolysis rates. In a second step, the coupled system was operated at recycling ratios (R) of 3 and 9. Fed C/N ratios of 0.58, 1.0 and 1.5 g C-formaldehyde g(-1) N-NH4(+) were used for every recycling ratio. The maximum nitrogen removal percentages were achieved at a C/N ratio of 1.0 g C-formaldehyde g(-1) N-NH4(+) for both recycling ratios. A fed C/N ratio of 1.5 g C-formaldehyde g(-1) N-NH4(+) caused a decrease in the efficiency of the system with respect to nitrogen removal, due to the presence of formaldehyde in the BAS reactor, which decreased the nitrification. Formaldehyde was completely removed in the BAS reactor and a heterotrophic layer formed around the nitrifying biofilm.     

Aerobic granular sludge in a sequencing batch reactor

In a laboratory scale sequencing batch reactor (SBR) granules were cultured under aerobic conditions. To enhance the growth of granular sludge the SBR was operated with very short sedimentation and draw phases resulting in the washout of slow settling biomass. Fast settling granules were retained in the reactor and thus had an advantage over flocs with a slower settling velocity. After 40 days of operation granules were the dominant form of microbial aggregates in the reactor, even though some pin-point flocs remained in the system. Granules taken from the reactor were stored for weeks without disintegrating. After about 130 days of operation the granule quality and COD-removal worsened. The reasons for that are yet to be investigated.     

Granulation in high-load denitrifying upflow sludge bed (USB) pulsed reactors

In this work, the effect of the application of a pulse system to anoxic upflow sludge bed (USB) denitrifying reactors for enhancing sludge granulation was studied. In all, three 0.8 L reactors (two operated with flow pulsation, P1 with effluent recycling and P2 without recycling, and one without pulsation and effluent recycling, no pulsation (NP)) were fed with a mixture of NaNO3 and glucose and inoculated with methanogenic granular sludge. The organic loading rate (OLR) and the nitrogen loading rate (NLR) were progressively increased and, at the end of the experiment, extremely high values were obtained (67.5 kgCOD/m3d and 11.25 kgN-NO3-/m3 d). Ammonia and nitrite accumulation in reactor NP were important in the maturation stage, decreasing the denitrification efficiency to 90%, while in reactor P1 only low nitrite values were obtained in the last few days of the experiment. In reactor P2, nitrogen removal was 100% most of the time. Several operational problems (flotation and the subsequent wash out of biomass) appeared in the NP reactor when working at high denitrifying loading rates, while in reactors P1 and P2 there were no notable problems, mainly due to the good characteristics of the sludge developed and the efficient degasification produced by the pulsing flow. The sludge formed in the NP reactor presented a flocculent structure and a total disintegration of the initial methanogenic granules occurred, while a small-sized granular biomass with a high specific density was developed in the pulsed reactors due to the shear stress produced.     

Role of mass transfer in overall substrate removal rate in a sequential aerobic sludge blanket reactor treating a non-inhibitory substrate

A laboratory study was undertaken to explore the role of mass transfer in overall substrate removal rate and the subsequent kinetic behavior in a glucose-fed sequential aerobic sludge blanket (SASB) reactor. At the organic loading rates (OLRs) of 2-8 kg chemical oxygen demand (COD)/m³-d, the SASB reactor removed over 98% of COD from wastewater. With an increase in OLR, the average granule diameter (d_p = 1.1-1.9 mm) and the specific oxygen utilization rate increased; whereas biomass density of granules and solids retention time decreased (13-32 d). The intrinsic and apparent kinetic parameters were evaluated using break-up and intact granules, respectively. The calculated COD removal efficiencies using the kinetic model (incorporating intrinsic kinetics) and empirical model (incorporating apparent kinetics) agreed well with the experimental results, implying that both models can properly describe the overall substrate removal rate in the SASB reactor. By applying the validated kinetic model, the calculated mass transfer parameter values and the simulated substrate concentration profiles in the granule showed that the overall substrate removal rate is intra-granular diffusion controlled. By varying different d_p within a range of 0.1-3.5 mm, the simulated COD removal efficiencies disclosed that the optimal granular size could be no greater than 2.5 mm.     

Optimization of the activated sludge anoxic reactor configuration as a means to control nutrient removal kinetically

Factors influencing the determination of optimum reactor configuration for activated sludge denitrification are investigated in this paper. A kinetic optimization method is presented to evaluate optimal pre- and post-denitrification bioreactor stages. Applying the method developed, simulation studies were carried out to investigate the impacts of the ratio of the influent readily biodegradable and slowly biodegradable substrates and the oxygen entering the denitrification zones on the optimal anoxic reactor configuration. In addition, the paper describes the effects of the slowly biodegradable substrate on the denitrification efficiency using external substrate dosing, and it demonstrates kinetic considerations concerning the hydrolysis process. It has been shown that as a function of the biodegradable substrate composition, the stage system design with three optimized reactor compartments can effectively increase reaction rates in the denitrification zones, and can provide flexibility for varying operation conditions.     

Floatation and control of granular sludge in a high-rate anammox reactor

The granule floatation is a serious issue of the anammox (anaerobic ammonium oxidation) process when high loading rates are applied that results in instability or even system collapse. The present study reports the granule floatation in an anammox reactor when high loading rates were applied. The comparison of enlarged photos taken for the settling and floating granules showed that the two kinds of granules both contained macroscopic gas pockets accounting for 11 ± 14% of total volume. The settling granules had gas tunnels that could release the gas bubbles, while the floating granules did not. The presence of gas bubbles enclosed in the gas pockets led to the small density of 979.2 ± 15.8 mg L⁻¹ and flotation of anammox granules. Consequently, the flotation caused washout of anammox granules and the deterioration of anammox process (volumetric removal rate decreased from 4.00 to 2.46 kg N m⁻³ d⁻¹). The collection of floating granules, breaking them into small pieces and then returning to the anammox reactor proved an effective control strategy. The volumetric removal rate was finally up to 16.5 kg N m⁻³ d⁻¹ after the control strategy was put into use.     

Fate of aniline and sulfanilic acid in UASB bioreactors under denitrifying conditions

Two upflow anaerobic sludge blanket (UASB) reactors were operated to investigate the fate of aromatic amines under denitrifying conditions. The feed consisted of synthetic wastewater containing aniline and/or sulfanilic acid and a mixture of volatile fatty acids (VFA) as the primary electron donors. Reactor 1 (R1) contained a stoichiometric concentration of nitrate and Reactor 2 (R2) a stoichiometric nitrate and nitrite mixture as terminal electron acceptors. The R1 results demonstrated that aniline could be degraded under denitrifying conditions while sulfanilic acid remains. The presence of nitrite in the influent of R2, caused a chemical reaction that led to immediate disappearance of both aromatic amines and the formation of an intense yellow coloured solution. HPLC analysis of the influent solution, revealed the emergence of three product peaks: the major one at retention time (R_t) 14.3 min and two minor at R_t 17.2 and 21.5 min. In the effluent, the intensity of the peaks at R_t 14.3 and 17.2 min was very low and of that at R_t 21.5 min increased (∼3-fold). Based on the mass spectrometry analysis, we propose the structures of some possible products, mainly azo compounds. Denitrification activity tests suggest that biomass needed to adapt to the new coloured compounds, but after a 3 days lag phase, activity is recovered and the final (N₂ + N₂O) is even higher than that of the control.     

Integrated removal of nitrate and carbon in an upflow anaerobic sludge blanket (UASB) reactor: Operating performance

Denitrification and methanogenesis of a synthetic wastewater containing volatile fatty acids and nitrate were obtained in a single-stage process using an upflow anaerobic sludge blanket (UASB) reactor. The reactor was initially inoculated with methanogenic granular sludge and was gradually adapted to nitrate by increasing the nitrate concentration in the influent. Excess carbon not utilized for denitrification was converted to methane. During steady-state at a loading of 336 mg NO₃-N/l/d (24 mmol NO₃/l/d) and 6600 mg COD/l/d more than 99% removal of both nitrate and carbon was achieved. Batch experiments with biomass from the reactor showed that approximately 90% of the added nitrate was recovered as nitrogen gas indicating that true dentrification occurred. This was further verified from mass balances over the reactor. The granules changed appearance during the first 5 months of operation being fluffy and buoyant, probably reflecting changes in the microbial composition induced by the presence of nitrate. However, during the next two months more dense granules with good settling abilities gradually established in the system making this kind of combined process feasible in a UASB reactor. Characterization of the produced granules showed that while the mean diameter and density was comparable to granules from purely methanogenic systems, although the strength was lower.     

Quantification of the shear stresses in a microbial granular sludge reactor

Since a certain level of hydrodynamic shear force is needed in the formation of microbial granules for wastewater treatment, a method for quantifying the shear stresses in a microbial granular sludge reactor is highly desirable. In this work a novel energy-dissipation-based model was established and validated to quantitatively describe the shear stresses in a granular sludge sequencing batch reactor (SBR). With this model, the shear stress at the solid–liquid interface in an SBR was estimated and the relative magnitudes of shear stresses induced by fluid, gas bubble and collision on granules were evaluated. The results demonstrate that the effect of reactor geometry on the global shear stress was significant. Both the shear stress at the microbial granule surface and the biomass-loss rate increased with an increase in biomass concentration in the SBR. The gas bubble and the collision were found to be the main source for the shear stress at the granule surface.     

Methanogenic granular sludge as a seed in an anaerobic baffled reactor

This paper reviews the importance of phase separation in anaerobic wastewater treatment, characteristics of anaerobic baffled reactor (ABR), properties of anaerobic granular sludge, granulation process and advantages of granular sludge over nongranular sludge. The performance data of ABR and its modified configurations with regard to carbonaceous matter removal by using various seed sludges are compared. It is concluded that enhanced wastewater treatment efficiencies can be achieved with methanogenic granular seed sludge in an ABR because of a number of advantages associated with granular biomass over nongranular aggregates.     

Anaerobic/oxic/anoxic granular sludge process as an effective nutrient removal process utilizing denitrifying polyphosphate-accumulating organisms

In a biological nutrient removal (BNR) process, the utilization of denitrifying polyphosphate-accumulating organisms (DNPAOs) has many advantages such as effective use of organic carbon substrates and low sludge production. As a suitable process for the utilization of DNPAOs in BNR, an anaerobic/oxic/anoxic granular sludge (AOAGS) process was proposed in this study. In spite of performing aeration for nitrifying bacteria, the AOAGS process can create anaerobic/anoxic conditions suitable for the cultivation of DNPAOs because anoxic zones exist inside the granular sludge in the oxic phase. Thus, DNPAOs can coexist with nitrifying bacteria in a single reactor. In addition, the usability of DNPAOs in the reactor can be improved by adding the anoxic phase after the oxic phase. These characteristics enable the AOAGS process to attain effective removal of both nitrogen and phosphorus. When acetate-based synthetic wastewater (COD: 600 mg/L, NH4-N: 60 mg/L, PO(4)-P: 10 mg/L) was supplied to a laboratory-scale sequencing batch reactor under the operation of anaerobic/oxic/anoxic cycles, granular sludge with a diameter of 500 microm was successfully formed within 1 month. Although the removal of both nitrogen and phosphorus was almost complete at the end of the oxic phase, a short anoxic period subsequent to the oxic phase was necessary for further removal of nitrogen and phosphorus. As a result, effluent concentrations of NH(4)-N, NO(x)-N and PO(4)-P were always lower than 1 mg/L. It was found that penetration depth of oxygen inside the granular sludge was approximately 100 microm by microsensor measurements. In addition, from the microbiological analysis by fluorescence in situ hybridization, existence depth of polyphosphate-accumulating organisms was further than the maximum oxygen penetration depth. The water quality data, oxygen profiles and microbial community structure demonstrated that DNPAOs inside the granular sludge may be responsible for denitrification in the oxic phase, which enables effective nutrient removal in the AOAGS process.     

Mechanisms and models for anaerobic granulation in upflow anaerobic sludge blanket reactor

Upflow anaerobic sludge blanket (UASB) reactor has been employed in industrial and municipal wastewater treatment for decades. However, the long start-up period required for the development of anaerobic granules seriously limits the application of this technology. In order to develop the strategy for rapid UASB start-up, the mechanisms for anaerobic granulation should be understood. This paper attempts to provide a up-to-date review on the existing mechanisms and models for anaerobic granulation in the UASB reactor, which include inert nuclei model, selection pressure model, multi-valence positive ion-bonding model, synthetic and natural polymer-bonding model, Capetown's model, spaghetti theory, syntrophic microcolony model, multi-layer model, secondary minimum adhesion model, local dehydration and hydrophobic interaction model, surface tension model, proton translocation-dehydration theory, cellular automaton model and cell-to-cell communication model. Based on those previous works, a general model for anaerobic granulation is also proposed. It is expected that this paper would be helpful for researchers to further develop a unified theory for anaerobic granulation and technology for expediting the formation of the UASB granules.