Bioresource Technology 151 (2014) 78–84
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Characteristics of aerobic granulation at mesophilic temperatures in wastewater treatment Fenghao Cui, Seyong Park, Moonil Kim ⇑ Department of Civil & Environmental Engineering, Hanyang University, 55 Hanyangdaehak-ro, Ansan City, Kyeonggido 426-791, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Aerobic granulation was carried out
at mesophilic temperatures (35 °C). Significant organic and ammonia
were simultaneously removed. Diversified species could be
developed through mesophilic aerobic granulation. The growth kinetics of heterotrophs and autotrophs were estimated. Biochemical and physical interactivities create a protective granule’s shell.
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 3 October 2013 Accepted 7 October 2013 Available online 22 October 2013 Keywords: Aerobic granulation Heterotrophs Autotrophs Growth kinetics Mesophilic temperatures
a b s t r a c t Compact and structurally stable aerobic granules were developed in a sequencing batch reactor (SBR) at mesophilic temperatures (35 °C). The morphological, biological and chemical characteristics of the aerobic granulation were investigated and a theoretical granulation mechanism was proposed according to the results of the investigation. The mature aerobic granules had compact structure, small size (mean diameter of 0.24 mm), excellent settleability and diverse microbial structures, and were effective for the removal of organics and nitrification. The growth kinetics demonstrated that the biomass growth depended on coexistence and interactions between heterotrophs and autotrophs in the granules. The functions of heterotrophs and autotrophs created a compact and secure layer on the outside of the granules, protecting the inside sludge containing environmentally sensitive and slow growing microorganisms. The mechanism and the reactor performance may promise feasibility and efficiency for treating industry effluents at mesophilic temperatures using aerobic granulation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Aerobic granulation is a process of microbial self-immobilization without the support of a carrier to form a multicellular association and compact structure (Beun et al., 1999; de Kreuk et al., 2005). The formation of granules in an aerobic sequencing batch reactor (SBR) has been extensively studied for high strength wastewater treatment (Adav et al., 2008; Abdullah et al., 2013). Wastewaters that require biological treatment at a high strength, like ⇑ Corresponding author. Tel.: +82 31 400 5142; fax: +82 31 502 5142. E-mail addresses: [email protected] (F. Cui), [email protected] (S. Park), [email protected] (M. Kim). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.025
pulp and paper effluents, many food processing effluents and anaerobic digested effluents, are often discharged at high temperatures (Pokhrel and Viraraghavan, 2004; Lefebvre and Moletta, 2006; Cui et al., 2011). The aerobic granulation appears to be a promising technology to treat these types of wastewaters, but has never been tested for simultaneous organics removal and nitrification at mesophilic temperatures (35 °C). Temperature can play a vital role in bacteria growth by influencing the rates of enzymatically catalyzed reactions and by affecting the rate of diffusion of substrate to the cells (Feller and Gerday, 2003). It has been reported that the applications of aerobic granulation at a thermophilic temperature (55 °C) have potential advantages over other applications, including low waste biomass
F. Cui et al. / Bioresource Technology 151 (2014) 78–84
production, higher degradation rates, elimination of cooling requirements for high temperature wastes, enhanced solubility and degradation of low-solubility substrates and rapid inactivation of pathogens (Zitomer et al., 2007). Despite this, thermophilic aerobic granulation may not be a practical approach due to the poor solubility of oxygen at such high temperatures. Detailed information of the mesophilic temperatures (30–40 °C) effect on aerobic granulation is limited. Furthermore, the formation of aerobic granules is a complicated ecological process, in which many factors need to be further investigated. The aerobic growth of heterotrophs and autotrophs could coexist and interact in aerobic granules (Yang et al., 2004). The heterotrophs are responsible for the oxidation of biodegradable organic carbon, whereas the autotrophs are responsible for the oxidation of ammonia to nitrite and nitrate through nitrification. In a conceptual microbial structure of the granules, a granule could simultaneously contain heterotrophs on the outside and autotrophs in the middle (Ni et al., 2008). In general, the aerobic granulation was carried out with the presence of high strength organic matters that creates competition between the autotrophs and heterotrophs for DO (Liu et al., 2003). The interspecies competition could result in a decrease in nitrification efficiency because the autotrophic growth rate is much lower than the heterotrophic growth rate (Nogueira et al., 2002). At mesophilic temperatures, the activated sludge appeared to have a negative impact on the performance of organics removal, whereas the nitrification proceeded better (Fdz-Polanco et al., 1994; Vogelaar et al., 2002). Therefore, the understanding of the growth and activity of autotrophs and heterotrophs in aerobic granules is important for optimizing the performance of wastewater treatment. This study demonstrated that a mesophilic (35 °C) aerobic granular biomass could be developed using the synthetic organic and ammonium wastewater. The process of mesophilic aerobic granulation was evaluated to understand the morphological, biological and chemical characteristics of granules. The roles of heterotrophs and autotrophs at mesophilic temperature were discussed based on experimental results and kinetic analysis. Furthermore, the theoretical mechanism for the formation of aerobic granules at a mesophilic temperature was discussed. 2. Methods
79
as NH4–N), KH2PO4 (70 mg L1), MgSO4 (20 mg L1), CaCl2 (100 mg L1), trace solution 1 (1 mg L1) and trace solution 2 (1 mg L1). Trace solution 1 contained (per liter deionized water) ethylenediaminetetraacetic acid (EDTA) (5 g) and FeSO47H2O (9.144 g). Trace solution 2 contained (per liter deionized water) EDTA (15 g), ZnSO47H2O (0.43 g), CoCl26H2O (0.24 g), MnCl2H2O (0.66 g), CuSO45H2O (0.25 g), NaMoO42H2O (0.22 g), NiCl26H2O (0.19 g), Na2SeO4H2O (0.21 g) and H3BO4 (0.014 g). 2.3. Analytical methods Chemical oxygen demand (COD), MLSS, mixed liquor volatile suspended solids (MLVSS) and sludge volume index (SVI) were determined according to Standard Methods (APHA, 2005). NH4– N was measured using a spectrophotometer (DR/2500, Method 8038, Nessler Method, Hach Co., USA). NO2–N and NO3–N were measured using an ion chromatography (790 Personal IC, Metrohm Ltd., Switzerland). The samples of effluent water were filtered by a 0.45 lm nylon syringe filter (Whatman International Ltd.) for the analysis of COD, NH4–N, NO2–N and NO3–N. Particle size distribution was analyzed by an electrophoretic light scattering system (Zeta-potential & Particle Size Analyzer ELSZ-2, Otsuka Electronic Co., Ltd., Japan). According to the methods proposed by Avcioglu et al. the oxygen uptake rate (OUR) was calculated by monitoring DO concentration (Avcioglu et al., 2003). The microstructure of mature granules was examined with a scanning electron microscope (SEM) (MIRA3, TESCAN Inc., USA). The chemical compositions of sludge were analyzed by an energy dispersive spectrometry system for the SEM (TEAMTM EDS, EDAX Inc., USA). 2.4. Determination of heterotrophs and autotrophs The biomass was removed from the SBR and placed into the batch reactors where it was aerated more than a week. During this period, oxygen uptake rate (OUR) and nitrate production rate (NPR) were periodically tested for determining the active biomass of heterotrophs (XH, mg L1) and autotrophs (XA, mg L1). In the OUR tests 20 mg L1 of thiourea was added to inhibit nitrification. The values of heterotrophic decay (bH, d1) and autotrophic decay (bA, d1) are obtained from the data on the change in OUR and NPR over time.
2.1. Operation of reactor
ln OUR ¼ lnðfd bH X H Þ bH t
ð1Þ
The laboratory SBR was a cylindrical acrylic glass vessel (100 cm in height and 6 cm in diameter), with a working volume of 2.85 L. Air was introduced through the bottom by a fine bubble aerator at 3 L min1. The temperature was controlled at around 35–37 °C by a water jacket. The reactor was operated in a successive cycle of 6 h, including 6 min of influent filling, 320–330 min of aeration, 20– 30 min of settling and 4 min of effluent discharge. The minimum DO concentration detected in the reactor was above 2 mg L1, while the pH fell in the range of 7.5–8.5. It was operated more than 70 days without excess sludge discharge; hence the effluent was the only passage for biomass wasting.
ln NPR ¼ lnðfd bA X A Þ bA t
ð2Þ
where fd is the biodegradable fraction of 0.8 (McCarty, 1975). The initial concentrations of active biomass in the batch reactors are determined by using the baseline endogenous OUR and NPR. Therefore, the initial XH and XA represent the active biomass of heterotrophs and autotrophs when the sludge is taken out of the SBR.
OURInitial ¼ fd bH X H;Initial
ð3Þ
NPRInitial ¼ fd bA X A;Initial
ð4Þ
2.2. Media 2.5. Estimation of growth kinetics The reactor was inoculated with activated sludge taken from an aeration tank of a wastewater treatment plant in Ansan City, Korea. The amount of inoculum was about 2.5 L, with a mixed liquor suspended solids (MLSS) concentration of 2500 mg L1. Synthetic wastewater prepared with tap water was used with glucose as a carbon source (450 ± 24 mg L1 as COD). The chemical addition to the synthetic wastewater included (NH4)2SO4 (150 ± 25 mg L1
The Gompertz (Eq. (5)) was adopted to describe the biomass growth of the aerobic granulation. The Gompertz model is regarded as one of the most appropriate models to describe bacteria growth data (Zwietering et al., 1990).
X H;A ¼ A expð expðRmax e ðk tÞ=A þ 1Þ
ð5Þ
80
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where A is the asymptote of the maximal value (mg L1), Rmax is the maximum biomass growth rate (mg L1 d1) and k is the lag time (d). A linear least square technique was used to estimate the biomass yield Y (H: heterotrophs, A: autotrophs, g COD g1 VSS) and the traditional decay coefficient b (Berthouex and Gan, 1991). The linearized equation is described as:
ðSIn SEff Þ=ðX H;A sÞ ¼ bH;A =Y H;A þ 1=Y H;A h
ð6Þ
where SIn is the influent substrate concentration (mg L1), SEff is the effluent substrate concentration (mg L1), s is the hydraulic retention time (HRT, hr) and h is the sludge retention time (SRT, d). In this study, the half-saturation coefficient and the maxim specific growth rate are adjusted based on the DO concentration profiles in the anoxic batch reactors. Both parameters are changed based on the simulation results to make the model fit the measured data.
dSO =dt ¼ ðð1 Y H Þ=Y H Þ lH ðSS =ðK S þ SS ÞÞ ðSO =ðK O;H þ SO ÞÞ X H ð7Þ dSO =dt ¼ ðð4:57 Y A Þ=YÞ lA ðSNH =ðK NH þ SNH ÞÞ ðSO =ðK O;A þ SO ÞÞ X A ð8Þ where SS is the soluble biodegradable organic concentration (mg COD L1), SNH is the soluble ammonia nitrogen concentration (mg NH4+–N L1), SO is the dissolved oxygen concentration (mg O2 L1), KS is the organic half-saturation coefficient for heterotrophs (mg L1), KNH is the ammonia half-saturation coefficient for autotrophs (mg L1), KO,H is the oxygen half-saturate coefficient for heterotrophs (0.2 mg L1), KO,A is the oxygen half-saturate coefficient for autotrophs (0.4 mg L1), lH is the maximum specific growth rate for heterotrophs (d1) and lA is the maximum specific growth rate for autotrophs (d1) (Henze et al., 2000). 3. Results and discussion 3.1. Organic removal and nitrification in mesophilic aerobic granulation The variations of chemicals (organic and nitrogen), biomass (MLVSS) and settleability (SVI) during the operation of SBR at mesophilic temperatures are shown Fig. 1a. It was shown that the concentrations of organics and ammonia were affected by the process of granulation. In order to discuss the relationship between the formation of granules and removal of organic matter and ammonia, the aerobic granulation process was divided into three phases according to reactor performance during different periods and continuous microscopic observation for changes in sludge shape. The three phases were biomass aggregation, aerobic granulation and steady state with mature granules. The aerobic granules appeared in the reactor after 20 days. After 30 days, SVI decreased to less than 80 mL g1, which is close to the typical SVI of matured granules (Peng et al., 1999). An extremely low SVI range, around 10– 20 mL g1, was observed and the size of aerobic granules gradually stabilized at a mean diameter of 0.24 mm after 40 days. The most characteristics of mature aerobic granules accorded with the studies by Tay et al. (2004) and Yang et al. (2005) which investigated the formation of aerobic granules at different organic loading rates and Nitrogen/COD ratios. The smaller size was probably due to the operation at low organic loading rate. Throughout the operation, the sludge was maintained in the reactor all along and little suspended flocs were washed out with discharge. As a result, the MLVSS increased up to 11 g L1 after 60 days. The effluent COD was stabilized within the concentration of less than 10 mg L1 (about 98% removal) after 45 days, and nitri-
fication started in the short term operation of 15 days. The ammonia removal showed high performance with an average removal efficiency of about 93%, meanwhile, high concentration of nitrate accumulated in the reactor. The particle size distribution demonstrated the difference between activated sludge and granular sludge (Fig. 1b). On operating day 50, a peak was clearly observed, with an abundance of granules that were 250 lm in diameter and a small amount of dense granules (0.1–0.3%) with a diameter of about 2000 lm. According to these results and microscopic observations, the mature granules that formed in this mesophilic cultivation were determined to be a highly compacted aggregation of biomass with high settleability. Fig 2 shows the comparative results of organic and nitrogen removal rates between aerobic flocs and aerobic granules. After the mature aerobic granules were developed in the reactor, the organic removal rate was increased from 0.75 kg COD m3 day1 to 0.97 kg COD m3 day1. The ammonium conversion rate was 0.15 kg N m3 day1 with aerobic flocs, and it was doubled to 0.32 kg N m3 day1 with aerobic granules. This means significant nitrifier biomass could be accumulated in the reactor by the mesophilic aerobic granulation. In addition, the nitrogen removal rate of aerobic granules increased compare with the aerobic flocs. Song et al. (2013) have reported that partial nitrification by ammoniaoxidizing bacteria and denitrification by heterotrophs can simultaneously occur on the surface of aerobic granules. Therefore, the nitrogen removal, beside nitrogen assimilation, could be carried out by heterotrophic denitrification with the aerobic granules. Besides substrate consumption and biomass production, bacteria release soluble microbial products (SMP) from substrate metabolism and biomass decay which could form the majority of the effluent COD (Barker and Stuckey, 1999). The organic substrate could be rapidly oxidized by aerobic heterotrophs, thus the SBR system includes feast and famine periods at sufficiently high HRTs. The COD was not completely removed, probably due to the SMP production by the biomass decay in famine period. In the famine period for organic substrate, the growth and accumulation of autotrophic nitrifiers could be enhanced at mosephilic temperatures. Consequently, the simultaneous organic removal and nitrification were successfully achieved in the SBR.
3.2. Sludge characteristics Microscopic observation showed that the shape of the mature granules was close to spherical, which was evidently different from the activated sludge floc. The microstructure of activated sludge and aerobic granules was observed using SEM. The activated sludge was entangled with large numbers of filamentous bacteria. Nevertheless, only a few filaments were found on the granules and they showed a compact structure with bacterial cells in the shapes of rods and cocci. It demonstrates that the microbial community of aerobic granules was completely different from the seed activated sludge. The mesophilic temperatures, the feed substrates (organic and ammonia) and the long SRTs could result in the development of diversified species. Elemental composition of the sludge was investigated for different biomass conditions in the reactor (Fig. 3). The activated sludge had higher element weights for carbon, nitrogen and oxygen, which is the main composition of the organisms compared to the granular sludge. This means that the granules were composed more of inorganic compounds. The salt elements that were mostly determined in the granules were sodium and calcium, which dramatically increased after granulation. In addition, phosphorus was also higher in the granules than in the activated sludge, which might indicate the presence of phosphate-accumulating organisms (PAOs) in the granules.
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Aerobic granulation
Steady state (mature granules)
(a) Chemicals, mg/L and SVI mL/g
120
Eff COD
Eff NH4-N
MLVSS SVI
Eff NO2-N Eff NO3-N
14
12
100
10
80
8
60
6
40
4
20
2
MLVSS, g/L
Biomass aggregation 140
0
0 0
10
20
30
40
50
60
70
Time, day 14
(b) Activated sludge (seeding) Matured aerobic granules (day 50)
Volume fraction, %
12
10
8
6
4
2
0 0
500
1000
1500
2000
2500
3000
Particle size, μm Fig. 1. Variations of chemicals, biomass and SVI (a) and particle size distributions of sludge (b).
3.3. Growth kinetics The growth kinetics of heterotrophs and autotrophs were investigated for the mesophilic aerobic granulation. The yield and decay values were determined using a conventional experimental method. The half-saturation coefficient and the maximum specific growth rate were estimated by the model calibration. Since bacteria grow exponentially, the Gompertz model was applied to predict and estimate the bacteria growth. Fig. 4 shows the results of kinetic analysis for the bacteria growth. The logistic and linear models statistically described the growth kinetics of microorganisms. The heterotrophs and autotrophs showed yield values of 0.6225 and 0.2443 g COD g1 VSS and decay values of 0.2450 and 0.0960 d1, respectively (Fig. 4a). After having calibration, the dissolved oxygen profiles were successfully predicted by the models (Eqs. (7) and (8)). The calibrated heterotrophic and autotrophic maximum specific growth rates were 3.18 and 1.52 d1, respectively, and the half-saturation coefficients were 22 and 1.57 mg L1, respectively (Fig. 4b). In the mesophilic aerobic granulation, it was estimated
that the heterotrophic growth was slower and the autotrophic growth was faster than the growth rates in conventional activated sludge process (lH = 6.0–13.0 d1, lA = 0.8–1.2 d1) (Henze et al., 2000; Rittmann and McCarty, 2001). The growth curve well modeled the measured heterotrophic and autotrophic biomass growth (Fig. 4c). The simulation results demonstrated that the biomass growth directly went through the exponential and the stationary phase without lag phase. The heterotrophs reach the maximum biomass concentration of 6920 mg L1 at day 140 and the autotrophs reach the maximum biomass concentration of 533 meg L1 at day 80. Thus, it was estimated that the biomass growth principally depends on heterotrophic populations. Microorganisms in the aerobic granule-based SBR experience feast and famine periods and hence the biomass growth rate could be confined by the starvation condition. In the simulation study by Zhou et al. (2013), the modified activated sludge model NO. 3 (ASM3) which described that the biomass uses readily biodegradable substrate for the simultaneous storage and growth process, and when readily biodegradable substrate is depleted under
F. Cui et al. / Bioresource Technology 151 (2014) 78–84
Aerobic flocs (day 0~20)
Aerobic granules (day 40~64) 0.35
1.2 Nitrogen removal rate 1.0
-3
Organic removal rate, kg COD m day
-1
Organic removal rate 0.30
Ammonium conversion rate 0.25
0.8 0.20 0.6 0.15 0.4 0.10 0.2
0.05
Nitrogen removal and -3 -1 ammonium conversion rates, kg N m day
82
0.00
0.0
Fig. 2. Average organic removal, nitrogen removal and ammonium conversion rates in the operation of aerobic flocs and aerobic granules.
50 Activated sludge (seeding) Aerobic granules at day 60
Element weight, %
40
30
20
10
0 s m m ur on ne en en ine cium um oru ulf rb yg luori diu esiu or og l ini l h r a S x o t h p i n m C S O Ca F C N g lu os a h A M P
Elements Fig. 3. Mass composition of each element for different sludge.
famine conditions, degradation of the storage polymers takes place. Finally, the biomass is subjected to decay and produce inert organic carbon (Zhou et al., 2013). In the reactor, a major portion of the biomass growth was based on organic oxidizing bacteria cells, which also assimilate ammonia for their growth and hold the absolute advantage for oxygen competition. The heterotrophs even obtain energy from internal microbial products to maintain growth during the famine period. When the reactor was undergoing limited conditions for organic substrate due to rapid consumption by heterotrophs, the ammonia oxidizing bacteria could predominantly function on the granules by using the remaining ammonia, whereas their flourishing time would not be too long because the ammonia for supporting growth was very limited. After the ammonia was completely nitrified, the autotrophs faced the famine time, the anoxic settling and discharging time and the heterotroph growing time due to the recharged organic substrate, which might
cause the rapid decay of autotrophs. As a result, the active heterotrophic biomass (about 50% in total biomass) and the active autotrophic biomass (about 2% in total biomass) did not sufficiently accumulated in the total biomass. 3.4. Theoretical mechanism of aerobic granulation An inchoate aggregation of biomass can be formed from preexisting flocs under intensive hydrodynamic shear force. Mature granules may be created by a diverse microbial community due to the existence of different zones, layers and chemical gradients in the granule. Accordingly, the size of the granule can influence the distribution of the microbial community. In this study, the microbial function at the oxygen-limited zone (anoxic and anaerobic) was neglected since most granules were small in size and growth of anaerobic microorganisms was extremely slow
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0.20
(a)
0.18 Heterotrophs (H) 0.16
Autotrophs (A) Slope=1/YA=4.0930 YA=0.2443
(SIn-SS)/(XH,Aτ)
0.14 0.12 0.10
Intercept=bA/YA=0.0164 bA=0.0040 hr-1 (0.0960 d-1)
0.08 0.06
Slope=1/YH=1.6065 YH=0.6225
0.04 Intercept=bH/YH=0.0164
0.02
bH=0.0102 hr-1 (0.2450 d-1)
0.00 0.00
0.01
0.02
0.03
0.04
0.05
1/θ, hr-1 6
(b)
Calibrated μ^A = 1.52 d-1
5
Dissolved oxygen, mg/L
Heterotroph Autotroph Model
Calibrated μ^H = 3.18 d-1 Calibrated KS = 22 mg L-1 Calibrated KNH= 1.57 mg L-1
4
XVSS = 1100 mg L-1 XH = 36 % XA = 2.2 % Initial COD = 50 mg L-1 Initial NH4-N = 50 mg L-1
3
2
1
0 0
2
4
6
8
10
12
14
16
18
20
Time, min 7000
700
(c)
AH = 6920 mg L-1
Heterotrophic biomass, mg/L
-1
AA = 533 mg L
5000
500
400
4000 Rmax,H = 89.42 mg L-1 d-1
300
3000
2000
200
Rmax,A = 12.17 mg L-1 d-1
Gompertz model
1000
Heterotrophs (H)
Autotrophic biomass, mg/L
600
6000
100
Autotrophs (A) 0
0 0
15
30
45
60
75
90
105
120
135
150
Time, day Fig. 4. Kinetic estimation for microbial growth in the aerobic granules.
compared with aerobic oxidizers. Both heterotrophs and autotrophs coexisted and interacted in the granules, and it is believed
that they played a vital role in developing the compact and stable granules because the great biomass was produced by them. Rapid
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growing heterotrophs are responsible for the enrichment of extracellular polymeric substrates (EPS), which play an essential role in maintaining the integrity and stability of the spatial structure in mature granules (Liu et al., 2004). Cations in the wastewater are also an important ionic cross-bridging for the negatively charged EPS involved in bacterial adhesion to a surface and accelerates aerobic granulation. The nitrifying populations have highly hydrophobic interactions, thus their growth at the high concentration ammonia and the optimal temperature (mesophilic) could contribute to the higher cell hydrophobicity (Kim et al., 2000). According to thermodynamic theory, the increase in cell surface hydrophobicity could cause a corresponding decrease in the excess Gibbs energy of the surface, which promotes cell-to-cell interaction. In conclusion, the functions of the heterotrophs and autotrophs created a compact and secure protective layer on the outside of the granules, that may have protected inside sludge containing environmentally sensitive and slow growing microorganisms such as anaerobes. Suspended, looser cells on the granules such as filamentous bacteria may have been grazed on by the protozoa and finally decayed to soluble or particulate organic matter. In addition, the biomass aggregation could be maintained by physical movements such as hydrodynamic force, diffusion force, gravity force and thermodynamic force, which accompanied microbial growth. As compared to conventional activated sludge process, the SBR system for aerobic granulation exhibited many benefits and advantages, such as simple in construction and operation; space saving and flexibility in scales; low sludge production and high treatment efficiency and so on. These are very attractive and it would be great practical and social importance if the mesophilic aerobic granulation based SBR system could be successfully implemented for wastewater processing. However, the start-up and the control of aerobic granulation are complex due to a lack of fundamental knowledge of the microbiology associated with the granulation process, which needs to be investigated for optimizing real wastewater treatment. Accordingly, the mechanism and reactor performance in this study indicated feasibility and efficiency for treating industry effluents at a mesophilic temperature using aerobic granulation. 4. Conclusion The mature aerobic granules were developed at 35 °C after operating the SBR reactor for 50 days showing that the heterotrophs and the nitrifying autotrophs were simultaneously enriched in the reactor. The possibility of diverse cultivation might be owing to the compact and protective outer shell of the granules formed by microbial, chemical and physical interactivities. The characteristics of aerobic granular sludge demonstrated that mesophilic aerobic granules based SBR process can be appropriate for combined treatment of high temperature effluent such as anaerobic-digested wastewater. Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1A2004633).
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Development and characterization of the partial nitrification aerobic granules in a sequencing batch airlift reactor. Bioresour. Technol. 139, 285–291. Tay, J.H., Pan, S., He, Y., Tay, S.T.L., 2004. Effect of organic loading rate on aerobic granulation. II: Characteristics of aerobic granules. J. Environ. Eng. 130 (10), 1102–1109. Vogelaar, J.C.T., Bouwhuis, E., Klapwijk, A., Spanjers, H., van Lier, J.B., 2002. Mesophilic and thermophilic activated sludge post-treatment of paper mill process water. Water Res. 36, 1869–1879. Yang, S.F., Tay, J.H., Liu, Y., 2004. Respirometric activities of heterotrophic and nitrifying populations in aerobic granules developed at different substrate N/ COD ratios. Curr. Microbiol. 49 (1), 42–96. Yang, S.F., Tay, J.H., Liu, Y., 2005. Effect of substrate nitrogen/chemical oxygen demand ratio on the formation of aerobic granules. J. Environ. Eng. 131 (1), 86– 92. Zhou, M., Gong, J., Yang, C., Pu, W., 2013. Simulation of the performance of aerobic granular sludge SBR using modified ASM3 model. Bioresour. Technol. 127, 473– 481. Zitomer, H.D., Duran, M., Alber, R., Guven, E., 2007. Thermophilic aerobic granular biomass for enhanced settleability. Water Res. 41, 819–825. Zwietering, M.H., Jongenburger, I., Rombouts, F.M., Van’t Riet, K., 1990. Modeling of the bacteria growth curve. Appl. Environ. Microbiol. 56 (6), 1875–1881.
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Characteristics of aerobic granulation at mesophilic temperatures in wastewater treatment Fenghao Cui, Seyong Park, Moonil Kim ⇑ Department of Civil & Environmental Engineering, Hanyang University, 55 Hanyangdaehak-ro, Ansan City, Kyeonggido 426-791, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Aerobic granulation was carried out
at mesophilic temperatures (35 °C). Significant organic and ammonia
were simultaneously removed. Diversified species could be
developed through mesophilic aerobic granulation. The growth kinetics of heterotrophs and autotrophs were estimated. Biochemical and physical interactivities create a protective granule’s shell.
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 3 October 2013 Accepted 7 October 2013 Available online 22 October 2013 Keywords: Aerobic granulation Heterotrophs Autotrophs Growth kinetics Mesophilic temperatures
a b s t r a c t Compact and structurally stable aerobic granules were developed in a sequencing batch reactor (SBR) at mesophilic temperatures (35 °C). The morphological, biological and chemical characteristics of the aerobic granulation were investigated and a theoretical granulation mechanism was proposed according to the results of the investigation. The mature aerobic granules had compact structure, small size (mean diameter of 0.24 mm), excellent settleability and diverse microbial structures, and were effective for the removal of organics and nitrification. The growth kinetics demonstrated that the biomass growth depended on coexistence and interactions between heterotrophs and autotrophs in the granules. The functions of heterotrophs and autotrophs created a compact and secure layer on the outside of the granules, protecting the inside sludge containing environmentally sensitive and slow growing microorganisms. The mechanism and the reactor performance may promise feasibility and efficiency for treating industry effluents at mesophilic temperatures using aerobic granulation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Aerobic granulation is a process of microbial self-immobilization without the support of a carrier to form a multicellular association and compact structure (Beun et al., 1999; de Kreuk et al., 2005). The formation of granules in an aerobic sequencing batch reactor (SBR) has been extensively studied for high strength wastewater treatment (Adav et al., 2008; Abdullah et al., 2013). Wastewaters that require biological treatment at a high strength, like ⇑ Corresponding author. Tel.: +82 31 400 5142; fax: +82 31 502 5142. E-mail addresses: [email protected] (F. Cui), [email protected] (S. Park), [email protected] (M. Kim). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.025
pulp and paper effluents, many food processing effluents and anaerobic digested effluents, are often discharged at high temperatures (Pokhrel and Viraraghavan, 2004; Lefebvre and Moletta, 2006; Cui et al., 2011). The aerobic granulation appears to be a promising technology to treat these types of wastewaters, but has never been tested for simultaneous organics removal and nitrification at mesophilic temperatures (35 °C). Temperature can play a vital role in bacteria growth by influencing the rates of enzymatically catalyzed reactions and by affecting the rate of diffusion of substrate to the cells (Feller and Gerday, 2003). It has been reported that the applications of aerobic granulation at a thermophilic temperature (55 °C) have potential advantages over other applications, including low waste biomass
F. Cui et al. / Bioresource Technology 151 (2014) 78–84
production, higher degradation rates, elimination of cooling requirements for high temperature wastes, enhanced solubility and degradation of low-solubility substrates and rapid inactivation of pathogens (Zitomer et al., 2007). Despite this, thermophilic aerobic granulation may not be a practical approach due to the poor solubility of oxygen at such high temperatures. Detailed information of the mesophilic temperatures (30–40 °C) effect on aerobic granulation is limited. Furthermore, the formation of aerobic granules is a complicated ecological process, in which many factors need to be further investigated. The aerobic growth of heterotrophs and autotrophs could coexist and interact in aerobic granules (Yang et al., 2004). The heterotrophs are responsible for the oxidation of biodegradable organic carbon, whereas the autotrophs are responsible for the oxidation of ammonia to nitrite and nitrate through nitrification. In a conceptual microbial structure of the granules, a granule could simultaneously contain heterotrophs on the outside and autotrophs in the middle (Ni et al., 2008). In general, the aerobic granulation was carried out with the presence of high strength organic matters that creates competition between the autotrophs and heterotrophs for DO (Liu et al., 2003). The interspecies competition could result in a decrease in nitrification efficiency because the autotrophic growth rate is much lower than the heterotrophic growth rate (Nogueira et al., 2002). At mesophilic temperatures, the activated sludge appeared to have a negative impact on the performance of organics removal, whereas the nitrification proceeded better (Fdz-Polanco et al., 1994; Vogelaar et al., 2002). Therefore, the understanding of the growth and activity of autotrophs and heterotrophs in aerobic granules is important for optimizing the performance of wastewater treatment. This study demonstrated that a mesophilic (35 °C) aerobic granular biomass could be developed using the synthetic organic and ammonium wastewater. The process of mesophilic aerobic granulation was evaluated to understand the morphological, biological and chemical characteristics of granules. The roles of heterotrophs and autotrophs at mesophilic temperature were discussed based on experimental results and kinetic analysis. Furthermore, the theoretical mechanism for the formation of aerobic granules at a mesophilic temperature was discussed. 2. Methods
79
as NH4–N), KH2PO4 (70 mg L1), MgSO4 (20 mg L1), CaCl2 (100 mg L1), trace solution 1 (1 mg L1) and trace solution 2 (1 mg L1). Trace solution 1 contained (per liter deionized water) ethylenediaminetetraacetic acid (EDTA) (5 g) and FeSO47H2O (9.144 g). Trace solution 2 contained (per liter deionized water) EDTA (15 g), ZnSO47H2O (0.43 g), CoCl26H2O (0.24 g), MnCl2H2O (0.66 g), CuSO45H2O (0.25 g), NaMoO42H2O (0.22 g), NiCl26H2O (0.19 g), Na2SeO4H2O (0.21 g) and H3BO4 (0.014 g). 2.3. Analytical methods Chemical oxygen demand (COD), MLSS, mixed liquor volatile suspended solids (MLVSS) and sludge volume index (SVI) were determined according to Standard Methods (APHA, 2005). NH4– N was measured using a spectrophotometer (DR/2500, Method 8038, Nessler Method, Hach Co., USA). NO2–N and NO3–N were measured using an ion chromatography (790 Personal IC, Metrohm Ltd., Switzerland). The samples of effluent water were filtered by a 0.45 lm nylon syringe filter (Whatman International Ltd.) for the analysis of COD, NH4–N, NO2–N and NO3–N. Particle size distribution was analyzed by an electrophoretic light scattering system (Zeta-potential & Particle Size Analyzer ELSZ-2, Otsuka Electronic Co., Ltd., Japan). According to the methods proposed by Avcioglu et al. the oxygen uptake rate (OUR) was calculated by monitoring DO concentration (Avcioglu et al., 2003). The microstructure of mature granules was examined with a scanning electron microscope (SEM) (MIRA3, TESCAN Inc., USA). The chemical compositions of sludge were analyzed by an energy dispersive spectrometry system for the SEM (TEAMTM EDS, EDAX Inc., USA). 2.4. Determination of heterotrophs and autotrophs The biomass was removed from the SBR and placed into the batch reactors where it was aerated more than a week. During this period, oxygen uptake rate (OUR) and nitrate production rate (NPR) were periodically tested for determining the active biomass of heterotrophs (XH, mg L1) and autotrophs (XA, mg L1). In the OUR tests 20 mg L1 of thiourea was added to inhibit nitrification. The values of heterotrophic decay (bH, d1) and autotrophic decay (bA, d1) are obtained from the data on the change in OUR and NPR over time.
2.1. Operation of reactor
ln OUR ¼ lnðfd bH X H Þ bH t
ð1Þ
The laboratory SBR was a cylindrical acrylic glass vessel (100 cm in height and 6 cm in diameter), with a working volume of 2.85 L. Air was introduced through the bottom by a fine bubble aerator at 3 L min1. The temperature was controlled at around 35–37 °C by a water jacket. The reactor was operated in a successive cycle of 6 h, including 6 min of influent filling, 320–330 min of aeration, 20– 30 min of settling and 4 min of effluent discharge. The minimum DO concentration detected in the reactor was above 2 mg L1, while the pH fell in the range of 7.5–8.5. It was operated more than 70 days without excess sludge discharge; hence the effluent was the only passage for biomass wasting.
ln NPR ¼ lnðfd bA X A Þ bA t
ð2Þ
where fd is the biodegradable fraction of 0.8 (McCarty, 1975). The initial concentrations of active biomass in the batch reactors are determined by using the baseline endogenous OUR and NPR. Therefore, the initial XH and XA represent the active biomass of heterotrophs and autotrophs when the sludge is taken out of the SBR.
OURInitial ¼ fd bH X H;Initial
ð3Þ
NPRInitial ¼ fd bA X A;Initial
ð4Þ
2.2. Media 2.5. Estimation of growth kinetics The reactor was inoculated with activated sludge taken from an aeration tank of a wastewater treatment plant in Ansan City, Korea. The amount of inoculum was about 2.5 L, with a mixed liquor suspended solids (MLSS) concentration of 2500 mg L1. Synthetic wastewater prepared with tap water was used with glucose as a carbon source (450 ± 24 mg L1 as COD). The chemical addition to the synthetic wastewater included (NH4)2SO4 (150 ± 25 mg L1
The Gompertz (Eq. (5)) was adopted to describe the biomass growth of the aerobic granulation. The Gompertz model is regarded as one of the most appropriate models to describe bacteria growth data (Zwietering et al., 1990).
X H;A ¼ A expð expðRmax e ðk tÞ=A þ 1Þ
ð5Þ
80
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where A is the asymptote of the maximal value (mg L1), Rmax is the maximum biomass growth rate (mg L1 d1) and k is the lag time (d). A linear least square technique was used to estimate the biomass yield Y (H: heterotrophs, A: autotrophs, g COD g1 VSS) and the traditional decay coefficient b (Berthouex and Gan, 1991). The linearized equation is described as:
ðSIn SEff Þ=ðX H;A sÞ ¼ bH;A =Y H;A þ 1=Y H;A h
ð6Þ
where SIn is the influent substrate concentration (mg L1), SEff is the effluent substrate concentration (mg L1), s is the hydraulic retention time (HRT, hr) and h is the sludge retention time (SRT, d). In this study, the half-saturation coefficient and the maxim specific growth rate are adjusted based on the DO concentration profiles in the anoxic batch reactors. Both parameters are changed based on the simulation results to make the model fit the measured data.
dSO =dt ¼ ðð1 Y H Þ=Y H Þ lH ðSS =ðK S þ SS ÞÞ ðSO =ðK O;H þ SO ÞÞ X H ð7Þ dSO =dt ¼ ðð4:57 Y A Þ=YÞ lA ðSNH =ðK NH þ SNH ÞÞ ðSO =ðK O;A þ SO ÞÞ X A ð8Þ where SS is the soluble biodegradable organic concentration (mg COD L1), SNH is the soluble ammonia nitrogen concentration (mg NH4+–N L1), SO is the dissolved oxygen concentration (mg O2 L1), KS is the organic half-saturation coefficient for heterotrophs (mg L1), KNH is the ammonia half-saturation coefficient for autotrophs (mg L1), KO,H is the oxygen half-saturate coefficient for heterotrophs (0.2 mg L1), KO,A is the oxygen half-saturate coefficient for autotrophs (0.4 mg L1), lH is the maximum specific growth rate for heterotrophs (d1) and lA is the maximum specific growth rate for autotrophs (d1) (Henze et al., 2000). 3. Results and discussion 3.1. Organic removal and nitrification in mesophilic aerobic granulation The variations of chemicals (organic and nitrogen), biomass (MLVSS) and settleability (SVI) during the operation of SBR at mesophilic temperatures are shown Fig. 1a. It was shown that the concentrations of organics and ammonia were affected by the process of granulation. In order to discuss the relationship between the formation of granules and removal of organic matter and ammonia, the aerobic granulation process was divided into three phases according to reactor performance during different periods and continuous microscopic observation for changes in sludge shape. The three phases were biomass aggregation, aerobic granulation and steady state with mature granules. The aerobic granules appeared in the reactor after 20 days. After 30 days, SVI decreased to less than 80 mL g1, which is close to the typical SVI of matured granules (Peng et al., 1999). An extremely low SVI range, around 10– 20 mL g1, was observed and the size of aerobic granules gradually stabilized at a mean diameter of 0.24 mm after 40 days. The most characteristics of mature aerobic granules accorded with the studies by Tay et al. (2004) and Yang et al. (2005) which investigated the formation of aerobic granules at different organic loading rates and Nitrogen/COD ratios. The smaller size was probably due to the operation at low organic loading rate. Throughout the operation, the sludge was maintained in the reactor all along and little suspended flocs were washed out with discharge. As a result, the MLVSS increased up to 11 g L1 after 60 days. The effluent COD was stabilized within the concentration of less than 10 mg L1 (about 98% removal) after 45 days, and nitri-
fication started in the short term operation of 15 days. The ammonia removal showed high performance with an average removal efficiency of about 93%, meanwhile, high concentration of nitrate accumulated in the reactor. The particle size distribution demonstrated the difference between activated sludge and granular sludge (Fig. 1b). On operating day 50, a peak was clearly observed, with an abundance of granules that were 250 lm in diameter and a small amount of dense granules (0.1–0.3%) with a diameter of about 2000 lm. According to these results and microscopic observations, the mature granules that formed in this mesophilic cultivation were determined to be a highly compacted aggregation of biomass with high settleability. Fig 2 shows the comparative results of organic and nitrogen removal rates between aerobic flocs and aerobic granules. After the mature aerobic granules were developed in the reactor, the organic removal rate was increased from 0.75 kg COD m3 day1 to 0.97 kg COD m3 day1. The ammonium conversion rate was 0.15 kg N m3 day1 with aerobic flocs, and it was doubled to 0.32 kg N m3 day1 with aerobic granules. This means significant nitrifier biomass could be accumulated in the reactor by the mesophilic aerobic granulation. In addition, the nitrogen removal rate of aerobic granules increased compare with the aerobic flocs. Song et al. (2013) have reported that partial nitrification by ammoniaoxidizing bacteria and denitrification by heterotrophs can simultaneously occur on the surface of aerobic granules. Therefore, the nitrogen removal, beside nitrogen assimilation, could be carried out by heterotrophic denitrification with the aerobic granules. Besides substrate consumption and biomass production, bacteria release soluble microbial products (SMP) from substrate metabolism and biomass decay which could form the majority of the effluent COD (Barker and Stuckey, 1999). The organic substrate could be rapidly oxidized by aerobic heterotrophs, thus the SBR system includes feast and famine periods at sufficiently high HRTs. The COD was not completely removed, probably due to the SMP production by the biomass decay in famine period. In the famine period for organic substrate, the growth and accumulation of autotrophic nitrifiers could be enhanced at mosephilic temperatures. Consequently, the simultaneous organic removal and nitrification were successfully achieved in the SBR.
3.2. Sludge characteristics Microscopic observation showed that the shape of the mature granules was close to spherical, which was evidently different from the activated sludge floc. The microstructure of activated sludge and aerobic granules was observed using SEM. The activated sludge was entangled with large numbers of filamentous bacteria. Nevertheless, only a few filaments were found on the granules and they showed a compact structure with bacterial cells in the shapes of rods and cocci. It demonstrates that the microbial community of aerobic granules was completely different from the seed activated sludge. The mesophilic temperatures, the feed substrates (organic and ammonia) and the long SRTs could result in the development of diversified species. Elemental composition of the sludge was investigated for different biomass conditions in the reactor (Fig. 3). The activated sludge had higher element weights for carbon, nitrogen and oxygen, which is the main composition of the organisms compared to the granular sludge. This means that the granules were composed more of inorganic compounds. The salt elements that were mostly determined in the granules were sodium and calcium, which dramatically increased after granulation. In addition, phosphorus was also higher in the granules than in the activated sludge, which might indicate the presence of phosphate-accumulating organisms (PAOs) in the granules.
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Aerobic granulation
Steady state (mature granules)
(a) Chemicals, mg/L and SVI mL/g
120
Eff COD
Eff NH4-N
MLVSS SVI
Eff NO2-N Eff NO3-N
14
12
100
10
80
8
60
6
40
4
20
2
MLVSS, g/L
Biomass aggregation 140
0
0 0
10
20
30
40
50
60
70
Time, day 14
(b) Activated sludge (seeding) Matured aerobic granules (day 50)
Volume fraction, %
12
10
8
6
4
2
0 0
500
1000
1500
2000
2500
3000
Particle size, μm Fig. 1. Variations of chemicals, biomass and SVI (a) and particle size distributions of sludge (b).
3.3. Growth kinetics The growth kinetics of heterotrophs and autotrophs were investigated for the mesophilic aerobic granulation. The yield and decay values were determined using a conventional experimental method. The half-saturation coefficient and the maximum specific growth rate were estimated by the model calibration. Since bacteria grow exponentially, the Gompertz model was applied to predict and estimate the bacteria growth. Fig. 4 shows the results of kinetic analysis for the bacteria growth. The logistic and linear models statistically described the growth kinetics of microorganisms. The heterotrophs and autotrophs showed yield values of 0.6225 and 0.2443 g COD g1 VSS and decay values of 0.2450 and 0.0960 d1, respectively (Fig. 4a). After having calibration, the dissolved oxygen profiles were successfully predicted by the models (Eqs. (7) and (8)). The calibrated heterotrophic and autotrophic maximum specific growth rates were 3.18 and 1.52 d1, respectively, and the half-saturation coefficients were 22 and 1.57 mg L1, respectively (Fig. 4b). In the mesophilic aerobic granulation, it was estimated
that the heterotrophic growth was slower and the autotrophic growth was faster than the growth rates in conventional activated sludge process (lH = 6.0–13.0 d1, lA = 0.8–1.2 d1) (Henze et al., 2000; Rittmann and McCarty, 2001). The growth curve well modeled the measured heterotrophic and autotrophic biomass growth (Fig. 4c). The simulation results demonstrated that the biomass growth directly went through the exponential and the stationary phase without lag phase. The heterotrophs reach the maximum biomass concentration of 6920 mg L1 at day 140 and the autotrophs reach the maximum biomass concentration of 533 meg L1 at day 80. Thus, it was estimated that the biomass growth principally depends on heterotrophic populations. Microorganisms in the aerobic granule-based SBR experience feast and famine periods and hence the biomass growth rate could be confined by the starvation condition. In the simulation study by Zhou et al. (2013), the modified activated sludge model NO. 3 (ASM3) which described that the biomass uses readily biodegradable substrate for the simultaneous storage and growth process, and when readily biodegradable substrate is depleted under
F. Cui et al. / Bioresource Technology 151 (2014) 78–84
Aerobic flocs (day 0~20)
Aerobic granules (day 40~64) 0.35
1.2 Nitrogen removal rate 1.0
-3
Organic removal rate, kg COD m day
-1
Organic removal rate 0.30
Ammonium conversion rate 0.25
0.8 0.20 0.6 0.15 0.4 0.10 0.2
0.05
Nitrogen removal and -3 -1 ammonium conversion rates, kg N m day
82
0.00
0.0
Fig. 2. Average organic removal, nitrogen removal and ammonium conversion rates in the operation of aerobic flocs and aerobic granules.
50 Activated sludge (seeding) Aerobic granules at day 60
Element weight, %
40
30
20
10
0 s m m ur on ne en en ine cium um oru ulf rb yg luori diu esiu or og l ini l h r a S x o t h p i n m C S O Ca F C N g lu os a h A M P
Elements Fig. 3. Mass composition of each element for different sludge.
famine conditions, degradation of the storage polymers takes place. Finally, the biomass is subjected to decay and produce inert organic carbon (Zhou et al., 2013). In the reactor, a major portion of the biomass growth was based on organic oxidizing bacteria cells, which also assimilate ammonia for their growth and hold the absolute advantage for oxygen competition. The heterotrophs even obtain energy from internal microbial products to maintain growth during the famine period. When the reactor was undergoing limited conditions for organic substrate due to rapid consumption by heterotrophs, the ammonia oxidizing bacteria could predominantly function on the granules by using the remaining ammonia, whereas their flourishing time would not be too long because the ammonia for supporting growth was very limited. After the ammonia was completely nitrified, the autotrophs faced the famine time, the anoxic settling and discharging time and the heterotroph growing time due to the recharged organic substrate, which might
cause the rapid decay of autotrophs. As a result, the active heterotrophic biomass (about 50% in total biomass) and the active autotrophic biomass (about 2% in total biomass) did not sufficiently accumulated in the total biomass. 3.4. Theoretical mechanism of aerobic granulation An inchoate aggregation of biomass can be formed from preexisting flocs under intensive hydrodynamic shear force. Mature granules may be created by a diverse microbial community due to the existence of different zones, layers and chemical gradients in the granule. Accordingly, the size of the granule can influence the distribution of the microbial community. In this study, the microbial function at the oxygen-limited zone (anoxic and anaerobic) was neglected since most granules were small in size and growth of anaerobic microorganisms was extremely slow
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0.20
(a)
0.18 Heterotrophs (H) 0.16
Autotrophs (A) Slope=1/YA=4.0930 YA=0.2443
(SIn-SS)/(XH,Aτ)
0.14 0.12 0.10
Intercept=bA/YA=0.0164 bA=0.0040 hr-1 (0.0960 d-1)
0.08 0.06
Slope=1/YH=1.6065 YH=0.6225
0.04 Intercept=bH/YH=0.0164
0.02
bH=0.0102 hr-1 (0.2450 d-1)
0.00 0.00
0.01
0.02
0.03
0.04
0.05
1/θ, hr-1 6
(b)
Calibrated μ^A = 1.52 d-1
5
Dissolved oxygen, mg/L
Heterotroph Autotroph Model
Calibrated μ^H = 3.18 d-1 Calibrated KS = 22 mg L-1 Calibrated KNH= 1.57 mg L-1
4
XVSS = 1100 mg L-1 XH = 36 % XA = 2.2 % Initial COD = 50 mg L-1 Initial NH4-N = 50 mg L-1
3
2
1
0 0
2
4
6
8
10
12
14
16
18
20
Time, min 7000
700
(c)
AH = 6920 mg L-1
Heterotrophic biomass, mg/L
-1
AA = 533 mg L
5000
500
400
4000 Rmax,H = 89.42 mg L-1 d-1
300
3000
2000
200
Rmax,A = 12.17 mg L-1 d-1
Gompertz model
1000
Heterotrophs (H)
Autotrophic biomass, mg/L
600
6000
100
Autotrophs (A) 0
0 0
15
30
45
60
75
90
105
120
135
150
Time, day Fig. 4. Kinetic estimation for microbial growth in the aerobic granules.
compared with aerobic oxidizers. Both heterotrophs and autotrophs coexisted and interacted in the granules, and it is believed
that they played a vital role in developing the compact and stable granules because the great biomass was produced by them. Rapid
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growing heterotrophs are responsible for the enrichment of extracellular polymeric substrates (EPS), which play an essential role in maintaining the integrity and stability of the spatial structure in mature granules (Liu et al., 2004). Cations in the wastewater are also an important ionic cross-bridging for the negatively charged EPS involved in bacterial adhesion to a surface and accelerates aerobic granulation. The nitrifying populations have highly hydrophobic interactions, thus their growth at the high concentration ammonia and the optimal temperature (mesophilic) could contribute to the higher cell hydrophobicity (Kim et al., 2000). According to thermodynamic theory, the increase in cell surface hydrophobicity could cause a corresponding decrease in the excess Gibbs energy of the surface, which promotes cell-to-cell interaction. In conclusion, the functions of the heterotrophs and autotrophs created a compact and secure protective layer on the outside of the granules, that may have protected inside sludge containing environmentally sensitive and slow growing microorganisms such as anaerobes. Suspended, looser cells on the granules such as filamentous bacteria may have been grazed on by the protozoa and finally decayed to soluble or particulate organic matter. In addition, the biomass aggregation could be maintained by physical movements such as hydrodynamic force, diffusion force, gravity force and thermodynamic force, which accompanied microbial growth. As compared to conventional activated sludge process, the SBR system for aerobic granulation exhibited many benefits and advantages, such as simple in construction and operation; space saving and flexibility in scales; low sludge production and high treatment efficiency and so on. These are very attractive and it would be great practical and social importance if the mesophilic aerobic granulation based SBR system could be successfully implemented for wastewater processing. However, the start-up and the control of aerobic granulation are complex due to a lack of fundamental knowledge of the microbiology associated with the granulation process, which needs to be investigated for optimizing real wastewater treatment. Accordingly, the mechanism and reactor performance in this study indicated feasibility and efficiency for treating industry effluents at a mesophilic temperature using aerobic granulation. 4. Conclusion The mature aerobic granules were developed at 35 °C after operating the SBR reactor for 50 days showing that the heterotrophs and the nitrifying autotrophs were simultaneously enriched in the reactor. The possibility of diverse cultivation might be owing to the compact and protective outer shell of the granules formed by microbial, chemical and physical interactivities. The characteristics of aerobic granular sludge demonstrated that mesophilic aerobic granules based SBR process can be appropriate for combined treatment of high temperature effluent such as anaerobic-digested wastewater. Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1A2004633).
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