Environ. Sci. Technol. 2005, 39, 2818-2827
Formation and Characterization of Aerobic Granules in a Sequencing Batch Reactor Treating Soybean-Processing Wastewater KUI-ZU SU AND HAN-QING YU* School of Chemistry, University of Science & Technology of China, Hefei, Anhui, 230026, China
Aerobic granules were cultivated in a sequencing batch reactor (SBR) fed with soybean-processing wastewater at 25 ( 1 °C and pH 7.0 ( 0.1. The granulation process was described via measuring the increase of sludge size. The formation of granules was found to be a four-phase process, that is, acclimating, shaping, developing, and maturated. A modified Logistic model could well fit with the granule growth by diameter and could be employed to estimate the maximum diameter, lag time, and specific diameter growth rate effectively. Both normal and lognormal distributions proved to be applicable to model the diameter distribution of the granules. The granulecontaining liquor was shear thinning, and their rheological characteristics could be described by using the HerschelBuckley equation. The suspended solids concentration, pH, temperature, diameter, settling velocity, specific gravity, and sludge volume index all had an effect on the apparent viscosity of the mixed liquor of granules. The matured granules had fractal nature with a fractal dimension of 1.87 ( 0.34. Moreover, 83% of matured granules were permeable with fluid collection efficiencies over 0.034. As compared to activated sludge flocs, the aerobic granules grown on the soybean-processing wastewater had better settling ability, mass transfer efficiency, and bioactivity.
Introduction The feasibility and efficiency of immobilization-based bioreactors for removing biodegradable organic matters have been investigated extensively, and upflow anaerobic sludge blanket (UASB) reactors with granules (1) and the Captor process with biofilm (2) have been widely used for wastewater treatment. In recent years, research attention has been turned toward developing aerobic granular sludge, which is generally known for its regular, dense, and strong microbial structure, good settling ability, high biomass retention, and the ability to withstand shock loading rate (3). It is expected that this new form of activate sludge, like the anaerobic granular sludge, could be employed for the treatment of municipal and industrial wastewaters. Aerobic granules have been cultivated in sequencing batch reactor (SBR) or sequencing batch airlift reactor fed with synthetic wastewaters composed of various substances, such as acetate (3), molasses (4), sucrose (5), and ethanol (6). According to the microscopic observation, the formation of * Corresponding author phone: +86 551 3607592; fax: +86 551 3601592; e-mail:
[email protected]. 2818
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aerobic granules grown on glucose and acetate was a gradual process from seed sludge to compact aggregates, further to granular sludge, and finally to matured granules (7). Previous studies demonstrated that operating conditions, such as a relatively short settling time, high superficial air velocity, low hydraulic retention time (HRT), high organic loading rate, and high ratio of height to diameter of the reactor, have been proved to benefit granulation (2, 3, 6-8). The characteristics of granular sludge in terms of settling velocity, size, shape, biomass density, hydrophobicity, physical strength, microbial activity, and extracellular polymeric substances (EPS) have been evaluated (3, 5, 7, 8). Although aerobic granulation has been investigated extensively, the granular growth course, however, has not been quantitatively evaluated. Thus, a unified and objective criterion to evaluate granulation process is still desired. Nonlinear curve simulation is an easy and efficient method to obtain the mathematical expression of the processes. Normal and log-normal distributions have been used to fit the size distribution of activated sludge flocs (9-11). Park and Ganczarczyk (12) employed the log-normal and normal distribution to evaluate the geometric characteristics of biomass washed out from an aerated submerged biological filter. Logistic model has been found to be effective for describing the growth of Lactobacillus helveticus (13), free and immobilized planktonic green algae (14), and Aspergillus niger (15). Rheology is a useful means for describing the formation of a body under the influence of stresses. Activated sludge suspensions are invariably non-Newtonian fluids, and the shear rate is nonlinearly related to the shear stress (16). The Herschel-Buckley equation has been used to model rheological characteristics (16):
τ ) τY + Kγ˘ m
(1)
where τ is shear stress, τY is yield stress, γ˘ is shear rate, and K and m are constants. The yield stress and apparent viscosity (ηa) of activated sludge flocs are dependent on their physicochemical properties, such as the suspended solids (SS) concentration, bound water, and surface charge (17, 18). The rheological characteristics of sludge can influence its stirring, settling, and mass transfer ability (16, 19, 20). However, there is no information about the rheological properties of aerobic granules. The fractal theory developed by Mandelbrot provides a systematic approach to the characterization of many natural and engineered systems that have no definite form or regularity, and this theory has been employed for characterizing bioparticles (21-23). The most important numerical parameter in fractal theory is the fractal dimension. Activated sludge flocs have proved to be fractal, and one-third of them are permeable through the settling experiments (21). The internal permeation of bioparticles can be revealed by the difference of observed and predicted settling velocities in water, while the fluid collection efficiency is an indication of whether the bioparticles are permeable (21, 22). From the settling velocities in water and denser solutions, mass transfer mechanisms of bioparticles can also be deduced to be molecular diffusion accompanied by limited convection (21, 23). In this study, the aerobic granules were cultivated in an SBR fed with soybean-processing wastewater, which is an industrial wastewater rich in proteins. The characteristics of bioparticles in granulation process were analyzed to give a quantitative description about it. Moreover, this study 10.1021/es048950y CCC: $30.25
2005 American Chemical Society Published on Web 02/23/2005
attempted to describe the bioparticle growth in the granulation process, and the rheological and fractal characteristics of the granules. It is expected that the information provided here would be useful for further understanding of the aerobic granulation as well as for its application for the treatment of municipal and industrial wastewaters.
Materials and Methods Reactor and Seed Sludge. The SBR reactor used in this study was similar to that described in a previous paper (5). The reactor had a working volume of 6.0 L with an internal diameter of 11.5 cm and a height of 80 cm. Effluent was drawn from the port at 30 cm from the bottom, resulting in 3.0 L of mixed liquor left in the reactor after effluent withdrawal. Air was introduced through an air diffuser by an air pump at the bottom of the reactor. The airflow rate was controlled via a gas-flow controller to keep the dissolved oxygen level over 1.5 mg/L in each aeration cycle. The seed sludge used for the reactor was taken from an aeration tank in Wangxiaoying Municipal Wastewater Treatment Plant, Hefei, China. The seed sludge had a mixed liquor suspended solids (MLSS) concentration of 12.6 g/L and a sludge volume index (SVI) of 74.2 mL/g. Its specific gravity and the settling velocity were 1.006 and 7.0 m/h, respectively. Sludge of 1.7 L was seeded to the SBR, resulting in an initial MLSS concentration of 5.4 g/L in the reactor. Wastewater Composition. The raw wastewater, containing soluble proteins of 5.5 ( 0.2 g/L and carbohydrates of 7.4 ( 0.2 g/L, was obtained from a local soybean-processing plant. It had pH value of 4.2 ( 0.6 and SS of 305 ( 17 mg/L. The soluble chemical oxygen demand (COD) and total nitrogen (TN) concentrations were 21.1 ( 2.6 g/L and 974 ( 112 mg/L, respectively. Because the wastewater contained sufficient amounts of nitrogen, only phosphorus as Na2HPO4 was added to ensure the ratio of COD to P to be 100:1 of the wastewater. In addition, the microelement solution of 1.0 mL/L was added, which contained (in mg/L): H3BO3, 50; ZnCl2, 50; CuCl2, 30; MnSO4‚H2O, 50; (NH4)6Mo7O24‚4H2O, 50; AlCl3, 50; CoCl2‚6H2O, 50; and NiCl2, 50. The influent pH value was adjusted to 7.0 ( 0.1 by the addition of NaHCO3 or HCl. The raw wastewater was diluted by 10 times using tap water to get the influent to the SBR. Operating Conditions. The reactor was operated sequentially as 5 min of influent filling, 220 min of aeration, 5 min of settling, and 10 min of effluent withdrawal. An air velocity of 0.4 m3/h was applied to the reactor, equivalent to a superficial upflow of 1.1 cm/s. The SBR was supplied with the influent COD concentration of around 2000 mg/L, corresponding to a loading rate of 6.0 kg COD/(m3d). The temperature of the reactor was maintained at 25 ( 1 °C using a belt heater and a temperature controller. Analytical Methods. Image Analysis. Microbial observation was conducted by using an optical microscope (Olympus CX41). The granule size was measured using an image analysis system (Image-pro Express 4.0, Media Cybernetics) with an Olympus CX41 microscope and a digital camera (Olympus C5050). With the imaging results, two parameters, that is, aspect ratio and roundness, could be calculated using the software (Image-pro Express 4.0, Media Cybernetics). Aspect ratio is defined as the ratio between the length of major axis and the length of minor axis of the ellipse equivalent to the object (i.e., an ellipse with the same area, first and second degree moments), as determined by major axis/minor axis. Roundness is determined by the following formula: (perimeter2)/ (4*π*area). Perimeter is the length of outer boundary of the object. Circular objects have a roundness ) 1, while other shapes have a roundness > 1. Specific Gravity. Sucrose was used to make a series of solutions with specific gravities of 1.005, 1.008, 1.011, 1.014,
1.017, 1.020, and 1.023. Ten granules were added into each of the eight 10-mL tubes filled with the sucrose solutions with different densities. Under quiescent conditions, the granules moved up or down in the tubes, depending on the solution density. In this way, the wet specific gravity of granules was measured. Hydrophobicity. The hydrophobic nature of the bioparticles was determined by measuring contact angle using axisymmetric drop shape analysis, according to the method proposed by Duncan-Hewitt et al. (24). A suspension of mixed liquor containing bioparticles was deposited on a cellulosic membrane filter. Samples were washed three times with deionized water, and residual water was removed by filtration. The drop shape of a sessile distilled water droplet placed on the layer of biomass was determined using a contact angle analyzer (Powereach JC2000A, Zhongchen Corp., Shanghai). Rheological Behavior. The sludge samples were concentrated by centrifugation at 3000 rpm for 5 min. The thickened sludge was then diluted to a predetermined concentration by using the supernatant. The apparent viscosity was measured after 1-min rotation using a multi-speed rotary viscometer (NDJ-4, Precise Science Instrument Corp., Shanghai) with rotating speeds of 0.3, 0.6, 1.5, 3, 6, 12, 30, and 60 rpm. Fractal Dimension and Permeability. The fractal dimension of the granules was determined following the methods proposed by Li and Yuan (21). The settling velocity was measured by recording the time taken for an individual granule to fall from a certain height in a measuring cylinder. The dry mass of granules was weighed with a balance (PerkinElmer, AD2B). Three columns with a diameter of 4.0 cm and a height of 40 cm were used to determine the settling velocities of each granule in water, EDTA, and NaCl solutions. The EDTA and NaCl solutions both had a density of 1.005 g/cm3. EPS. The extraction of EPS from the activated sludge followed the procedure described by Sponza (25). Sludge was harvested by centrifugation at 12 000 rpm for 15 min and washed with 0.9% NaCl solution twice prior to extraction. It was suspended in 25 mL of 2% (m/V) EDTA solution. The sludge suspension was incubated overnight at 4 °C. Each sample was centrifuged at 12 000 rpm for 15 min. The resulting supernatant of the extraction were dialyzed using a membrane of 8000 molecular weight cut off against ultrapure water at 4 °C for 2 days. The carbohydrate concentration in EPS was determined as glucose equivalent using the Dubois method (26), whereas the protein concentration was measured as bovine albumin equivalent using the Lowry method (27). Other Analyses. Measurement of COD, MLSS, mixed liquor volatile suspended solids (MLVSS), SVI, and specific oxygen uptake rate (SOUR) were performed using the standard methods (28).
Results Performance of the Reactor. In the initial operating days, the influent COD was stepwise increased from 500 to 2000 mg/L (Figure 1A), and the loading rate was correspondingly increased from 1.5 to 6.0 kg COD/(m3 d) at day 17 (Figure 1B). The reactor performance was improving continuously in terms of COD removal efficiency during the operation (Figure 1C). This improvement became obvious after around 7 days of operation, and the COD removal efficiency was kept at 98-99% afterward. A settling period of 10 min was applied in the first week to prevent severe wash out of sludge and was then decreased to 5 min in the subsequent one week. As a result of the improvement of settling ability, MLSS kept increasing despite the excess sludge discharge, even after the settling period was reduced to 5 min on day 15. The sludge properties, such as the MLVSS/MLSS and MLSS, are shown in Figure 1D and VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Calculated Parameters in Eq 2a 1.24 ( 0.10 39.97 ( 1.80 0.12 ( 0.02
Dmax (mm) t0 (day) k (1/day) a
R2 ) 0.988.
bioparticles kept increasing (Figure 2D and E). At day 46, granules had a mean diameter of about 0.9 mm (Figure 2F). Afterward, granules kept growing in a much lower speed up to the end of the operation (Figure 2G and H). Figure 3 illustrates the changes of mean diameter, settling velocity, specific gravity, and SVI of the bioparticles in the granulation process. The formation of granules from seed sludge was a gradual process, as evidenced by the increase of mean diameter of the granules (Figure 3A). Figure 3B shows that the settling velocity of the bioparticles increased swiftly from 8.9 m/h at day 24 to 33.2 m/h at day 46. As illustrated in Figure 3C, the specific gravity of activated sludge increased after granulation. It was 1.006 g/cm3 at the beginning and increased to 1.020 g/cm3 after the granules were formed at day 43. The SVI remained almost unchanged in the initial 7 days, and then decreased from 41 mL/g at day 7 to 17 mL/g at day 33 (Figure 3D). A modified Logistic model (13) is employed for describing the granulation process by diameter:
D(t) )
FIGURE 1. Operating parameters and performance of the SBR: (A) influent COD; (B) loading rate; (C) COD removal efficiency; (D) MLVSS/ MLSS; and (E) MLSS. E, respectively. After day 25, the MLSS was stabilized at 8.011.0 g/L, which was much higher than that of a conventional activated sludge treatment system. The ratio of MLVSS to MLSS increased continuously from 78% of seed sludge to 89% of granules. The high MLSS/MLVSS ratio might be attributed to the fact that the inorganic salts concentration in the soybean-processing wastewater was less than the actual municipal wastewater. Characterization of the Granulation Process. Images of the sludge in the granulation process are shown in Figure 2. Seed sludge had a fluffy, irregular, and loose-structure morphology (Figure 2A). The color of activated sludge gradually changed from brown to yellow with the process of experiment (Figure 2B). After 2-weeks of operation, small granules with diameters of 0.1-0.3 mm were observed (Figure 2C). Thereafter, the number and average diameter of the 2820
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Dmax
(2)
1 + e-k(t-t0)
where t is the operating time (days), D(t) is the mean diameter of bioparticles on day t (mm), Dmax is the asymptote of the curve, that is, the maximum mean diameter of bioparticles (mm), k is the specific growth rate by diameter (1/day), and t0 is the lag time (day). A regression was performed with the experimental data using the program Microsoft Origin 6.0 (Figure 3A), and the results are listed in Table 1. As shown in Figure 3A, the granulation process was well simulated by the modified Logistic model with a high correlation coefficient of 0.988. A lag time of around 40 days was calculated for the granulation process, whereas the maximum mean diameter of matured granules and specific growth rate by diameter were estimated to be 1.24 mm and 0.12 1/day, respectively. These values were in good agreement with the observed values (Table 1). Profiles of Granule Size Distribution. Both normal (eq 3) and log-normal distributions (eq 4) have been used for fitting the size distribution of activated sludge flocs (9-11). In this work, the two models were employed to simulate the diameter distribution of granules after their formation.
y)
y)
A1
e-(x-xc1) /2w1 w1x2π A2
2
2
e-(lnx-xc2) /2w2 w2x2π
(3)
2
2
(4)
where x is the diameter, y is the frequency of diameter x, xc1 and xc2 are the mathematical expectations, w1 and w2 are variances, and A1 and A2 are constants. As illustrated by Figure 4, the diameter distribution of granules was well described with the two distribution curves. The correlation coefficients were 0.972 for the normal distribution and 0.975 for the log-normal distribution, respectively. This implies that, with an increase in granule size, the diameters of granules turned into more concentrated around the average value from dispersed ones.
FIGURE 2. Images of sludge in the granulation process: (A) seed sludge; (B) sludge at day 8; (C) sludge at day 15; (D) sludge at day 21; (E) sludge at day 43; (F) sludge at day 46; (G) sludge at day 54; and (H) sludge at day 60. Physicochemical Characteristics of Granules. At the end of the 60-day operation, aerobic granules were taken from the SBR for the measurement of their physicochemical characteristics. The granules were spherical or elliptical in shape and yellow in color, and their surface was covered with plenty of microorganisms such as vorticella (Figure 2H). As shown in Table 2, the mean contact angle decreased substantially from 40.5° for seed sludge to 19.3° for the matured granules. The carbohydrate content in EPS remained almost unchanged, while the protein concentration increased from 2.2 to 4.1 mg/g SS. The SOUR of the bioparticles increased from 11.0 mg/L O2/(g SS h) for the seed sludge to 17.8 mg/L O2/(g SS h) for the granules on day 60. This suggests that the granules had a higher bioactivity than the seed sludge. Rheological Characteristics of the Granules. The granular sludge samples, GS-1, GS-2, and GS-3 with different mean
TABLE 2. Characteristics of the Seed Sludge and Granular Sludge at Day 60 diameter (mm) SVI (mL/g) settling velocity (m/h) specific gravity SOUR (mg/L O2/(g SS h)) contact angle (deg) carbohydrates in EPS (mg/g SS) proteins in EPS (mg/g SS)
seed sludge
granular sludge
0.10 ( 0.05 74.2 ( 5.7 7.0 ( 1.3 1.006 ( 0.001 11.0 ( 0.6 40.5 ( 5.3 3.2 ( 0.2
1.22 ( 0.85 30.8 ( 5.3 36.6 ( 8.8 1.017 ( 0.001 17.8 ( 2.2 19.3 ( 3.2 3.1 ( 0.5
2.2 ( 0.6
4.1 ( 0.8
diameters of 0.376, 0.550, and 1.228 mm, were taken from the SBR reactor at the end of the 60-day operation. Figure VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Relationship between shear stress and shear rate for the suspensions of granular sludge with different mean diameters: (A) GS-3; (B) GS-1; and (C) GS-2.
FIGURE 3. Sludge characteristics in the granulation process: (A) mean diameter; (B) settling velocity; (C) specific gravity; and (D) SVI.
FIGURE 6. Apparent viscosity as a function of: (A) shear rate (SS ) 20 g/L); and (B) shear time (SS ) 18 g/L, rotational speed of 12 rpm) for GS-2 at temperature 10 °C and pH 7.1. shear time, and the apparent viscosity were established as follows:
ηa ) 376.31 + 10601.86 exp(-γ˘ /0.52) + 2610.31 exp(-γ˘ /4.19) (5) FIGURE 4. Measured and calculated diameter distribution of granules.
ηa ) 156.05 + 57.48 exp(-t/12.98)
5 illustrates the correlation of the shear stress and shear rate for their suspensions. The Herschel-Buckley equation (eq 1) was used to model the changes of shear stress, and it fit well with the observed results with high correlation coefficients (R2 ) 0.992, 0.999, and 0.973). This suggests that the granular sludge samples behaved in a non-Newtonian fashion and did exhibit a yield stress. As shown in Figure 6, the apparent viscosity (ηa) decayed exponentially with an increase in shear rate (γ˘ ) and shear time (t). It implies that the suspension of the granular sludge was shear thinning. The relationships between the shear rate,
Figure 7 shows the apparent viscosity as a function of SS concentration, temperature, and pH. The apparent viscosity of granules was affected by these and several other factors, for example, SVI, settling velocity, and specific gravity. Suspensions of different granules with identical SS concentration were tested at the same temperature and pH, and the results are shown in Table 3. The apparent viscosity increased with increasing mean diameter, settling velocity, and SVI, whereas it decreased with increasing specific gravity. Settling Velocities and Permeabilities of Granules. A total of 48 aerobic granules with the diameter of 0.9-2.5 mm were
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(6)
TABLE 3. Characteristics of Three Granule Samples mean diameter (mm) SVI (mL/g) specific gravity settling velocity (m/h) apparent viscositya (mPa s) a
GS-1
GS-2
GS-3
0.376 27.8 ( 2.3 1.020 ( 0.001 13.7 ( 4.0 54.6 ( 4.5
0.550 27.7 ( 2.0 1.018 ( 0.001 15.6 ( 2.1 53.0 ( 3.8
1.228 36.9 ( 4.2 1.014 ( 0.001 23.3 ( 5.5 150.0 ( 8.0
Temperature 25 °C, SS ) 25 g/L.
FIGURE 8. Fractal and porous characteristics of granules: (A) relationship of diameter and dry mass after log-log transformation; and (B) porosity in relation to granule diameter.
FIGURE 7. Apparent viscosity as a function of: (A) SS (temperature 25 °C, pH 7.1); (B) temperature (SS ) 22 g/L, pH 7.1); and (C) pH (SS ) 20 g/L, temperature 10 °C) for GS-2 at a rotational speed of 60 rpm. analyzed to determine their fractal characteristics and permeabilities. Based on the slope of the logarithmic relationship between the dry mass (Wd) and size (d), as illustrated in Figure 8A, the fractal dimension D of the aerobic granules was calculated as 1.87 ( 0.34. The porosity increased from 0.965 to 0.999 with an increase in diameter from 0.9 to 2.5 mm, indicating that larger granules were more porous (Figure 8B). According to Li and Yuan (21), a bioparticle permitting only 3.4% of the fluid to flow through it could not be considered as permeable. As shown in Figure 9C, 83% of the granules tested in this study were permeable, which was much higher than the activated sludge flocs with only 36% permeable ones reported by Li and Yuan (21). The high permeability of the aerobic granules suggests that they were likely to have a higher mass transfer efficiency than that of the activated sludge flocs. The porous structure of the aerobic granules was able to facilitate the passage of oxygen and substrates as well as the release of metabolic products. The fractal dimension of the aerobic granules was 1.87 ( 0.34, indicating the fractal features of aerobic granules. The
ratio between the observed and predicted settling velocities was larger than unity, suggesting that the internal permeation of the granules was sufficient to influence their ability to settle (29). This further confirms that the aerobic granules were permeable. The granules had settling velocities of 0.41-1.27 cm/s in water, which were much larger than that (0.17-0.42 cm/s) of activated sludge flocs (21). As shown in Figure 9A, the slope of the settling velocity related to the size was 0.32, slightly larger than that predicted by Stokes’ law for porous but impermeable granules of 0.30 (21), suggesting that the aerobic granules were porous and permeable. The dimensionless ratios between the observed and predicted settling velocities (Γ) varied from 0.72 to 4.06, with an average of 1.51, implying that the internal permeation of the granules did affect their settling velocities (Figure 9B). The fluid collection efficiencies of the granules were in the range of 0.005-0.720 with an average of 0.260 (Figure 9C). The ratio of the observed and predicted settling velocities as well as the fluid collection efficiency did not change significantly with the diameter of granules. When the granules settled from water into a slightly denser solution of NaCl or EDTA, the settling velocities of the granules were significantly reduced in the higher density solution, as compared to those in water (Figure 10A). The ratios of the settling velocities in the denser NaCl solution to those in water varied from 0.52 to 1.03, while the ratios between the settling velocities in the denser EDTA solution and in water were in the range of 0.38-1.07 (Figure 10B). Their settling VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 10. Settling experiments in water, EDTA, and NaCl: (A) settling velocities in three solutions (4, water; b, NaCl; ", EDTA); and (B) ratios of the settling velocities in NaCl and EDTA solutions to those in water (4, theoretical prediction; b, NaCl/water; ", EDTA/ water).
FIGURE 9. Settling experiment in water: (A) settling velocity; (B) ratio of observed and predicted settling velocities; and (C) fluid collection efficiency in relation to granule diameter. velocities in EDTA solution were slightly lower than those in NaCl, because its larger size and greater molecular weight led to a lower diffusivity in granules.
Discussion In all previous studies concerning aerobic granulation, various synthetic wastewaters rich in carbohydrates (4-6) or fatty acids (3) have been used as a carbon source. So far, no aerobic granulation with an actual industrial wastewater has been reported yet. In the present work, aerobic granules were cultivated in an SBR treating soybean-processing wastewater at 25 ( 1 °C. This wastewater was rich in proteins with a ratio of protein:COD of 0.26:1. When a UASB reactor is used to treat protein-laden wastewaters, anaerobic sludge granulation has not been readily achieved (1). The presence of a high concentration of protein in wastewater, for example, dairy wastewater, has a significant negative effect on the formation of anaerobic granules. In this work, however, with the wastewater rich in proteins the aerobic granules were cultivated and became mature within 46 days. The granulation time was similar to those with carbohydrate-rich wastewater (4-6). This implies that the substrate component was not a crucial factor in the granulation of activated sludge. The information provided here would be useful for the development of aerobic-granule-based bioreactors for the treatment of industrial wastewaters. The aerobic granules grown on soybean-processing wastewater had excellent settling ability and high bioactivity. As illustrated in Figure 3, both settling velocity and specific gravity of the sludge increased in the granulation process. The average settling velocities of the aerobic granules were comparable to those of anaerobic granules in UASB (1) and 2824
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are at least 3 times greater than those of activated sludge flocs (lower than 9 m/h) (8). The significant improvement of specific gravity of the sludge indicates the granules had a highly dense and compact structure. This benefits for the solid-liquid separation and biomass retaining in the reactor. The mean diameter of matured granules was 1.22 ( 0.85 mm in this work. This value was comparable to results of many other studies on aerobic granules, for example, 0.30.5 mm from Peng et al. (30), and 1.0-1.3 mm from Jang et al. (31). However, it was substantially smaller than 3.3 mm reported by Beun et al. (6) and 1.9-2.3 mm reported by Tay et al. (3). Granule size depends on many factors, including substrate characteristics, reactor configuration, solids retention time, HRT, organic loading rate, pH, temperature, etc. A large amount of work has demonstrated that anaerobic granules grown on protein-rich wastes are much smaller than carbohydrate-rich ones (1). The soybean-processing wastewater was rich in proteins. This might be partially responsible for the smaller granule size in this study, as compared to that of granules grown on carbohydrate-laden wastewaters. Another possible reason for the small granule size could be associated with the high shear force applied on the SBR in this present work. These warrant further investigations. An increase in size of bioparticles did not necessarily result in a lower bioactivity, as evidenced by an increase in SOUR of sludge after the granulation (Table 2). For aerobic granules, oxygen transfer limitation did not occur for the radius smaller than the oxygen penetration depth of about 0.7 mm as measured with microelectrode, even when the oxygen concentration in bulk liquid was as low as 2 mg/L (31). This observation might be useful for explaining the increase in SOUR during the granulation. One possible reason for the increase of SOUR [in unit of mg/L O2/(g SS h)] was the increase in active microorganisms in the sludge, evidenced as an increase in MLVSS/MLSS from 78% to 89% in Figure 1D. The SS concentration, rather than VSS concentration, was used for the calculation of SOUR. The high shear force might also be partially responsible for the increase of SOUR. As reported
by Tay et al. (3), a high shear force stimulated the respiration activities of aerobic granules in a significant manner, resulting in an increase of 50% in SOUR when the superficial air velocity increased from 0.3 to 3.6 cm/s. The aerobic granulation may be initiated by microbial self-adhesion, resulting from the physical collision to trigger bacterium-to-bacterium contact (32). Multicell contacts are then evolved into stable cell aggregations through production of EPS and other environment-induced genetic effects that facilitate the cell-cell interaction (1). Shaping of the threedimensional structure of microbial aggregates is driven by hydrodynamic shear forces. In steady state, EPS can mediate both cohesion and adhesion of cells and play a crucial role in maintaining the structural integrity in a community of immobilized cells. The amount of EPS in sludge is affected by its growing conditions and wastewater composition. The EPS composition is of importance for granulation of sludge due to the influence of EPS on the surface charge and energy, which have much effect on adsorption and adherence (33). Moreover, EPS can cause the hydration of granule surfaces and protect granules against the shear stress and attachment to gas bubbles, favoring the stability of granules (1). The EPS in anaerobic granules consists mainly of proteins and carbohydrates, typically in a ratio of 2:1-6:1 (33). In the present study, the protein-to-carbohydrate ratio of EPS, lower than 1 for the flocs, almost doubled for the granules, implying that protein content of EPS became more abundant than carbohydrates during granulation. Such differences in the EPS content and the protein-to-carbohydrate ratio are probably instrumental with respect to the aerobic granulation mechanisms. The increase in protein content of EPS enhanced neighbor microbial cells and formed a cross-linked network by attraction of organic or inorganic materials (32). This observation is in accord with those of anaerobic granules reported by Schmidt and Ahring (33, 34). In the initial stage of the operation, bioparticles became denser under high shear force, mainly due to the bridging process among EPS, microbial cells, and ions (32). With a decrease in settling time, light flocs were washed out and denser bioparticles remained in the SBR. Subsequently, aerobic granulation was accompanied with the growth of aerobic microorganisms, and their attachment and detachment processes. This was a shaping and developing process. In the end, a balance was reached, and a steady-state operation was approximated. According to the analysis above and the observation of bioparticles, the aerobic granulation process could be categorized into four phases, that is, acclimating, shaping, developing, and matured (Figure 3A). This four-phase process is divided by the three turning points, granules initiated, shaped, and matured. The state in which small, irregular granules were being developed is defined as “granules initiated”. The corresponding period from the inoculation to the granules initiated is called the acclimating period. In this period, flocs in seed sludge congregated and formed denser and irregular aggregates. Some small granules appeared in this phase, and the settling velocity and specific gravity of the sludge did not change significantly. The shaping phase is defined as the process of change from dense and irregular shaped aggregates to regular granules. Size and the settling velocity of the sludge increased slowly in this period, while the specific gravity improved significantly, indicating that the change of settling velocity lagged behind that of specific gravity. The developing phase describes the conversion process from the shaped granules to matured ones. Granules in this period grew drastically with a specific growth rate by diameter of 0.12 1/day (Table 1). The matured phase is defined as the period after the granules became matured when the specific growth rate by diameter declined, and the
settling velocity and specific gravity were kept at a relatively stable value. The Logistic model (eq 2) was found to be appropriate for quantitatively describing the granulation process. With this equation, the lag time was estimated as 40 days, which covered the acclimating and shaping periods as the granulation progressed (Figure 3A). The specific growth rate by diameter (k) and the maximum mean diameter (Dmax) of granules calculated from this equation were both in accord with the observed ones (Table 1). These three parameters can be used as indexes to describe the granulation process quantitatively. The evolvement of diameter during the granulation process was found to be a sigmoid with lag time, a rapidly growing phase followed by a plateau phase. This implies that the granulation of sludge was initiated by compaction of flocs to obtain denser bioparticles, rather than a significant increase in size. Cell surface hydrophobicity, in terms of contact angle, was found to decrease in this study, and this was opposite from the results of others (3, 5, 32). This might result from the difference in microbial species, operating conditions, and the nonstandardized measuring procedures. Flow curves of all of the granular sludge samples fit well to the Herschel-Buckley equation (eq 1). It suggests that the aerobic granules were viscoplastic materials, which began to flow only when a yield stress exceeded a certain value. This might be associated with the stronger interconnected network among the granules. Like flocculent sludge, granular sludge was also shearthinning. At a certain SS concentration, granular sludge with a smaller SVI and a greater specific gravity had a smaller apparent viscosity (Table 3). This linked to the volume fraction of the granules in the suspension (φ) and can be predicted by the Einstein equation (7):
ηa ) ηo(1 + 2.5φ)
(7)
where ηo is solvent viscosity. As the volume fraction of granular sludge with a smaller SVI or a greater specific gravity became greater, they had larger apparent viscosities. Apparent viscosity increased exponentially with the SS concentration (Figure 7A). This was in good agreement with the results for activated sludge flocs (16). As shown in Figure 9C, the fluid collection efficiency of the granules was in the range of 0.005-0.540, lower than that of flocs generated from latex microspheres (21). This suggests that EPS secreted by the aerobic microorganisms might clog the pores within granules, thus reducing their permeabilities, although the granules were porous and fractal. The ratios of settling velocities of the granules in the denser solution and water were larger than those theoretical predictions about rapid completion of mass transfer throughout the granules, suggesting that a certain amount of water could have been enclosed and remained within the pores of a granule. This resulted in a slower settling velocity in the denser liquid than was predicted. However, it does not imply that there was a significant convective flow through the granules (35). Although both solutions of NaCl and EDTA had identical densities and viscosities, the granules had more reduction in settling velocity when they settled in EDTA solution than in NaCl solution. This is due to the large difference in molecular weight and size between the two chemicals; that is, NaCl has a much higher diffusivity in water than does EDTA molecules. Therefore, NaCl diffused into the granules at a rate considerably faster than that for EDTA. These results suggest that molecular diffusion might play an important role in the mass transfer through the aerobic granules. This is in accord with the mass transport model widely applied for biological treatment systems (36). VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 11. Morphology of sludge during the granulation process: (A) roundness; and (B) aspect ratio. The morphology is an important index for characterizing bioparticles (6). The morphology of sludge in terms of roundness and aspect ratio in the process of granulation is shown in Figure 11. The values of roundness and aspect ratio of circular objects are equal to 1.00, while the values of those with other shapes are beyond one. That is, objects with a shape close to circle have a roundness and aspect ratio with values close to 1.00. As illustrated by Figure 11, the roundness and aspect ratio of sludge all decreased to 1.00 with the formation of granules, suggesting that the flocs were gradually evolving into round granules. This was mainly induced by the detachment of loosely adhered microorganisms on the granule surface under high shear forces. The roundness and aspect ratios of the matured granules were comparable to those of Beun et al. (6).
Acknowledgments We wish to thank the NSFC-RGC (Grant No. 50418009), and the Trans-Century Training Program Foundation for Young Talents, Ministry of Education, China, for the partial support of this study.
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Received for review July 9, 2004. Revised manuscript received December 19, 2004. Accepted January 20, 2005. ES048950Y
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