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Partial nitrification to nitrite for treating ammonium-rich organic wastewater by immobilized biomass system
Bioresource Technology 100 (2009) 2341–2347
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Partial nitrification to nitrite for treating ammonium-rich organic wastewater by immobilized biomass system J. Yan, Y.Y. Hu * School of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 23 July 2008 Received in revised form 19 November 2008 Accepted 20 November 2008 Available online 6 January 2009 Keywords: Ammonium-rich organic wastewater Partial nitrification Degradation of organics SA immobilized biomass beads Anammox
a b s t r a c t This study focused on the characteristics of the partial nitrification and degradation of organics with immobilized biomass beads in the treatment of ammonium-rich organic wastewater. Sodium alginate (SA) was selected as the best entrapment support after comparing partial nitrification rate and adsorption efficiency. The immobilization methods were optimized by an orthogonal experiment. Zeta position and BET surface area were used to explain the adsorption behavior of SA immobilized beads. FT-IR revealed that a SA immobilized biomass bead was not a simply physical mixture of SA and biomass. The porous structure of SA immobilized biomass beads were observed by scanning electron microscopy (SEM), which confirmed the porosity of the beads. According to the experimental data, the effects of pH and temperature on partial nitrification and COD removal were evidently weakened in SA immobilized biomass beads due to the ‘‘protective” effect of immobilization, whereas the effects of HRT and DO were enhanced. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Nitrogen has become a major problem in water quality management. Biological nitrification–denitrification is the most commonly used process for nitrogen removal from wastewater, especially municipal wastewater (Ruiz et al., 2003). However, these systems also have disadvantages, such as high operational costs of the processes because of the oxygen and organic carbon requirements. Compared to conventional nitrification and denitrification process, over 50% less oxygen demand and absence of organic carbon addition offer considerable cost savings in the combined partial nitrification/Anammox process (Fux et al., 2002). Some nitrogenous wastewater have high organic content. Since ammonia-oxidizing bacteria (AOB) might live with aerobic heterotrophic bacteria, partial nitrification could take place in combination with degradation of organic carbons (Yamamoto et al., 2008). Therefore, the combined partial nitrification/Anammox process might be a promising new method for the treatment of influents with high organic carbons and ammonium concentration. The growth rate of autotrophic AOB is lower than that of heterotrophic bacteria, with which they have to compete for oxygen, and without long retention times the suspended nitrifiers will be easily washed out of the reactor. The biomass concentration is increased by recirculation of the sludge after sedimentation, but limited by the efficiency of the sedimentation vessel. Besides, the ammonia oxidation rate is strongly influenced by the nature of nitrifying cul* Corresponding author. Tel.: +86 20 39380506; fax: +86 20 39380569. E-mail address: [email protected] (Y.Y. Hu). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.11.038
tures and a variety of environmental factors, including substrate concentration, dissolved oxygen (DO), temperature and pH. However, immobilization techniques can be used to overcome these problems. Immobilization is an efficient method to prevent biomass from being washed out and allow hyperconcentrated cultures. They can lead to relatively small reactors, provide some protection from adverse temperatures and toxic shocks, which would help in maintaining year-round treatment (Morita et al., 2007). Immobilized biomass can be divided into ‘‘naturally” attached biomass (biofilm) and ‘‘artificially” immobilized biomass. Biofilm has been widely applied in wastewater treatment. However, some particles can become anaerobic in the centre and settle to the reactor floor. One of the most common techniques for artificially immobilization is gel entrapment. Both natural and synthetic polymers can be used as the immobilization support, but it must fulfill various requirements, such as photo-transparency, non-toxicity, retention of cellular viability, and stability in the culture medium (Mallick, 2002). This immobilization technique is commonly used to immobilize a pure strain of bacteria because the mechanisms of pure strains are easy to understand (Fierro et al., 2008; Hill et al., 2008; Shieh and Tsao, 2004; Benyahia and Polomarkaki, 2005). Nevertheless, the immobilization of activated sludge has also been reported (Rostron et al., 2001; Seo et al., 2001; Isaka et al., 2007). Compared to pure strain of bacteria, immobilization of activated sludge could remove multiple pollutants due to the biodiversity of the activated sludge. This study focuses on the characteristics of the partial nitrification and degradation of organics with an immobilized biomass in
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treating ammonium-rich organic wastewater. It serves as a first step in the Anammox process with partial denitrification via nitrite. After four materials, i.e. sodium carboxymethylcellulose (Na-CMC), sodium alginate (SA), polyvinyl alcohol and sodium alginate (PVA–SA), and chitosan (CTS), were prepared as supports for entrapping the biomass, the best entrapment support were selected by comparing partial nitrification rate and adsorption efficiency. Then, the best immobilization method was obtained by orthogonal experiment and the physicochemical characteristics of immobilized beads were investigated. Finally, the hydraulic retention time (HRT), pH, temperature and dissolved oxygen (DO) tolerance for immobilized beads were studied. The objective of this work was to determine if immobilization has advantages in reactor operation when pH, DO and temperature were variable under ammonia and COD loading applied. 2. Methods 2.1. Immobilization methods 2.1.1. Reagents The immobilization supports, Na-CMC (high viscosity; 2.0% in H2O at 25 °C: 300–600 mPa s; content of sodium: 6.5–6.8%), were supplied by Fuchen Chemical Enterprise Co. in Tianjin, China. Sodium alginate (SA; viscosity: 150 mPa s) was chemically pure grade. Polyvinyl alcohol (PVA) was analytically pure grade, and CTS (degree of deacetylation: 75%) was obtained from Boao Biotechnology Ltd. (Shanghai, China). 2.1.2. Acclimation of biomass Biomass were obtained from the activated sludge of the aeration tank of an urban wastewater treatment plant (WWTP) in Guangzhou, China, and acclimated with synthetic ammonia and COD feeding for two month in two 5 L sequencing batch reactor (SBR) and a volume exchange ratio of about 0.5. Both reactors were provided with a thermostatic jacket and temperature were maintained at 30 ± 1 °C using thermostatic bath. Both reactors were mechanically stirrered (JB300-D, Biaoben, Chia). Two pumps (Beta/4a 0708, Prominent, Germany) and oxygen valves were necessary to operate the reactor. The influent was maintained at 4 °C in a 10 L tank. Both reactors were drawn by gravity discharge using an electro-valve. Dissolved oxygen (DO) was measured with a electrode (550A, YSI, USA) and controlled at 0.5 mg L1 by adjusting the air flow rate manually. The pH was measured with a electrode (pH 6, Ecoscan, Singapore) and adjusted to about 7.5 by addition of acid (H2SO4 1 M) or alkali (NaOH 1 M) solution, respectively. The composition of the feeding solution was shown in Table 1, the trace solution was according to Strous et al. (1998). Ammonia, nitrite, nitrate and COD concentrations were measured everyday. 2.1.3. Preparation of immobilization beads The preparation method of immobilized beads was described as follow. The acclimated biomass was washed by distilled water three times before use. The biomass was concentrated by centrifuge at 1000 rpm for 5 min. One portion of the concentrated
Table 1 Composition of feeding solution. Parameter
g L1
Parameter
g L1
NH4Cl CaCl2 KH2PO4 MgSO4 FeSO4 7H2O
1.383 0.30 0.07 0.02 0.009
NaHCO3 EDTA Traces solution C2H12O6
2.16 0.006 1.25 mL L1 0.156
biomass (28.8 g VSSL1) was mixed thoroughly with one portion of the support solution as shown in Table 2. This mixture was then pumped (7524-55, Cole-Parmer Instrument, USA) into kieselgel tube with pinhead for drop formation (Pure support solution was pumped into kieselgel tube for preparing plain immobilized beads) (Seo et al., 2001). The extruded drops fell into a crosslink solution to form solid beads. The beads were transferred to a stabilization solution with aeration at 30 °C, the stabilization solution was prepared by mixing the cross linking agent with the feeding solution as shown in Table 1, and the pH of the stabilization solution was
Table 2 Immobilization condition for four kinds of beads. Support aqueous solution (w/v)
Crosslink solution (w/v)
Crosslink time (h)
SA (5%) PVA (10%)-SA (2%) Na-CMC (2%) CTS (3%)a
4% CaCl2 4% CaCl2 21.6% FeCl3 1% Polyphosphoric acid (PPA)
12 12 1 6
a The chitosan solution was prepared by mixing chitosan flakes with 5% acetic acid.
Table 3 Operation condition for each reactor at different periods. Study at constant DO = 4 mg L1, pH 7.5, T = 30 °C
Study at constant pH 7.5, T = 30 °C
Period A-support study
Period B-support absorption efficiency study
No.
Support
No.
Support
1 2 3
SA bead CTS bead CMC bead
1 2 3
Plain SA bead Plain CTS bead Plain CMC bead Plain PVA–SA bead
4
PVA–SA bead Study at constant T = 30 °C, DO = 4 mg L1, pH 7.5 Period D-HRT study
4 Study at constant T = 30 °C, DO = 4 mg L1, HRT = 30 h Period E-pH study
No.
HRT (h)
No.
pH
1 2 3 4 5 6 7 8 9
2.5 5.0 7.5 10.0 12.5 15.5 20.0 30.0 35.0
1 2 3 4 5 6 7 8 9 10
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
Study at constant DO = 4 mg L1, pH 8, HRT = 30 h
Study at constant T = 30 °C, pH 8, HRT = 30 h
Period F-temperature study
Period G-DO study
No.
T (°C)
No.
DO (mg L1)
1 2 3 4 5 6
15 20 25 30 35 40
1 2 3 4 5 6 7 8 9
1.8 2.1 3.2 3.4 3.9 4.2 4.6 5.1 5.2
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adjusted to about 7.5. At last, removed and rinsed the beads with distilled water three times. Additional activity recovery time was not necessary in this method (Kim et al., 2000).
Electrophoretic mobilities of particles were measured by the instrument and converted to zeta potential by using Smoluchowski equation.
2.2. Experimental method and procedure
2.4. Surface area and porosity
The batch experiments were carried out in 10 SBRs of 1 L and a volume exchange ratio of about 0.8. A 200 mL of immobilized beads was added to each reactor (20% reactor fill). All the reactors were magnetic stirred. A PVC net was installed at the bottom of the reactor (pore size 1 mm) to keep the immobilized beads away from the rotor. Other parts of the experimental system were operated as described above (Section 2.1.2.) The experimental time covered 7 periods. The performance of each reactor was different in each period. A summary of the operating conditions for each reactor is given in Table 3. The composition of the feeding solution shown in Table 1 is during the experiment except period B and C. During period B, it was done with one litre of feed with NHþ 4 -N concentration of 1 . Period C 1000 mg L1 and NO 2 -N concentration of 550 mg L was the orthogonal experiment for optimizing the immobilization method, and the orthogonal experiment with three levels and four factors were designed. The four factors were the concentration of support solution, mixing volume ratio of support solution and concentrated biomass, the concentration of cross linking agent and cross linking time. The three levels of each factor are showed in Table 4. The arrangement of orthogonal experiment was listed in Table 5.
Adsorption–desorption isotherms 77 K of both immobilized biomass and plain beads were determined using a computer controlled micromeritics ASAP 2020M (USA) apparatus. The specific surface area and pore size were determined by application of the Brunauer, Emmett and Teller (BET) method to the adsorption data (Ambrogi et al., 2008).
2.3. Zeta position A sample of 2 g immobilized SA bead was added to 100 mL deionized water and then mixed at 1500 rpm for 24 h by magnetic stirrer (ML-902, Shanghai suhao Instrument Ltd., China) kept in biochemical incubator (SPX-150B-Z, Shanghai China) at 30 ± 0.5 °C to obtain the suspension. Zeta potential measurements of the suspension obtained were carried out by Nano-ZS model (3800, Malvern Instrument Ltd., UK) zetasizer instrument. Table 4 Factors and levels of orthogonal experiment. No.
SA% (w/v)
SA:concentrated biomass (v/v)
CaCl2 (w/v)
Cross linking time (h)
1 2 3
5 7 10
1:1 1:1.5 1:2
2 4 8
12 24 48
Table 5 Analysis of crosstab data.
2.5. Infrared spectroscopy FT-IR spectroscopy was conducted on the immobilized biomass and plain beads. The immobilized beads were fully washed with deionized water, dried and ground to powder. Then, the powder was mixed with KBr and analyzed by an FT-IR spectrometer (Vector 33, Bruker Instrument Ltd., Germany). The experiments were run with air as the background, and the resolution and number of scans were 4.0 cm1 and 32, respectively. 2.6. Scanning electron microscopy Scanning electron microscopy (SEM) was used to study the cross-section morphology of the immobilized beads and to compare the surface characteristic among free biomass, immobilized biomass beads and plain beads. A ESEM (XL-30, Philips Instrument Ltd., Netherlands) was used for the specimens at 10 kV. 2.7. Analytical methods Volatile suspended solids (VSS), chemical oxygen demand (COD), ammonium ðNHþ 4 -NÞ, nitrites ðNO2 -NÞ and nitrates ðNO3 -NÞ were measured according to standard methods (APHA, 1995). The volume of immobilized bead was measured by draining method, which measured the volume of mixed liquor displaced when immobilized beads were added. The average wet density of immobilized bead was calculated by the average wet quality and volume. Concentrations of free ammonia (FA) and free nitrous acid (FNA) were calculated as a function of pH, temperature and total ammonium as nitrogen (TAN), for FA, or total nitrite (TNO2), for FNA (Ganiué et al., 2007):
FA ðmg NL1 Þ ¼
No.
SA% (w/v)
SA:concentrated biomass (v/v)
CaCl2 (w/v)
Cross linking time (h)
Concentration nitrite (mg L1)
1 2 3 4 5 6 7 8 9 k1 k2 k3 Rang
5% 7% 10% 5% 7% 10% 5% 7% 10% 202.26 191.22 191.00 11.26
1:1 1:1 1:1 1:1.5 1:1.5 1:1.5 1:2 1:2 1:2 140.44 195.12 248.92 108.48
4% 2% 8% 2% 8% 4% 8% 4% 2% 197.00 168.78 220.7 53.92
48 12 24 24 48 12 12 24 48 225.74 217.02 141.72 84.02
22.24 56.64 61.56 75.64 54.76 64.72 104.38 79.82 64.72
Note: Rang = kmaxkmin, k: sum of each level, concentrations of nitrite were average values.
TAN
1 þ ð10pH =K NH e Þ TNO2 1 FNA ðmg NL Þ ¼ pH 1 þ ðK NO Þ e =10
ð1Þ ð2Þ
¼ e6344=ð273þTÞ K NH e
ð3Þ
K NO e
ð4Þ
2300=ð273þTÞ
¼e
The activation energy of a reaction can be determined graphically by taking the natural logarithm of Arrhenius equation which describes the relationship between the reaction rate and the temperature, as shown in the following equation. Where A is the frequency factor for the reaction, R is the universal gas constant, T is the temperature (K), and Ea is the activation energy.
ln K ¼
Ea þ ln A RT
ð5Þ
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3. Results and discussion
3.3. Optimizing the SA immobilization method
3.1. Partial nitrification by different supporting immobilized beads
Based on the discussion above, SA might be an excellent support for partial nitrification. In order to obtain the optimal immobilization method, an orthogonal experiment was carried out. The arrangements of orthogonal experiments were listed in Table 5, which including nine sets of experiments, the result was the concentration of nitrite when HRT was 30 h. The last four rows of Table 5 show the results of different factors affecting on the partial nitrification processes by SA immobilized beads. The most significant effect among the four factors on partial nitrification was found to be the mixing volume ratio of support solution and concentrated biomass, followed by the concentration of cross linking agent, cross linking time and the concentration of support solution. The change of the concentration of support solution did not show significant effect on the partial nitrification, so the concentration of support solution of 5% was recommended for the immobilization process. The concentration of nitrite in the effluent of the reactor increased conspicuously with the increase of the mixing volume ratio of support solution and concentrated biomass, it means that the optimal mixing ratio could be adjusted to 1:2 in order to achieve a higher nitrite accumulation rate. A further increase of the concentration of cross linking agent may also improve partial nitrification efficiency. On the contrary, concentration of nitrite in the effluent of the reactor decreased significantly with the increase in cross linking time, so the cross linking time of 12 h was recommended. From the study mentioned above, the optimum immobilization methods were determined, which were: mixing volume ratio of support solution concentrated biomass of 1:2, concentration of cross linking agent of 8% , cross linking time of 12 h and concentration of support solution of 5%.
Nitrite production by SA immobilized bead was the highest, the concentration of nitrite was 28.85 mg L1 and 105.71 mg L1 when HRT was 15 h and 30 h, respectively. CMC immobilized bead showed the lowest partial nitrification efficiency, the concentration of nitrite was 0.25 mg L1 and 2.40 mg L1 when HRT was 15 h and 30 h, respectively. The order of partial nitrification efficiencies were SA > PVA–SA > CTS > CMC. (For PVA–SA immobilized bead, the concentration of nitrite was 22.04 mg L1 and 76.73 mg L1 when HRT was 15 h and 30 h, respectively. For CTS immobilized bead, the concentration of nitrite was 1.5 mg L1 and 29.9 mg L1 when HRT was 15 h and 30 h, respectively.) No nitrate was detected during the whole experiment. There were similar phenomena observed in nitrification systems by four immobilized beads that the sum of the dissolved nitrogen compounds in the effluent was lower compared with the inlet ammonium concentration. The most serious nitrogen loss occurred (about 84.2%) in CMC immobilized bead system. It suggested that adsorption of nitrogen by immobilized beads might occur in these systems. 3.2. Adsorption behavior of plain gel beads It was found that all the four plain gel beads could adsorb ammonia, as shown in Fig. 1. SA plain gel beads had 10.9% and 12.6% removal in 22 h and 40 h, respectively. Adsorption efficiency by CTS plain gel beads was also relatively high (9.4% and 9.5% in 22 h and 40 h, respectively). The order of adsorption of ammonium was SA > CTS > CMC > PVA. Fig. 1 also showed the adsorption of nitrite by four plain gel beads. CTS plain gel beads had significant 13.3% and 28.0% removal in 22 h and 40 h, respectively. Adsorption efficiency by CMC plain gel beads was also relatively high (13.9% and 22.1% in 22 h and 40 h, respectively). This result could explain the extraordinary low nitrite accumulation in CTS and CMC immobilized beads system. The order of adsorption of nitrite was CTS > CMC > SA > PVA. Since nitrite is the oxidation production of ammonia-oxidizing bacteria (AOB), the aim of partial nitrification is to obtain the nitrite accumulation. Thus, the adsorption of nitrite should be minimized in order to obtain the nitrite accumulation, and the adsorption of ammonia was less important compared with nitrite.
Fig. 1. Adsorption efficiency of ammonium and nitrate by plain gel beads with time.
3.4. Characteristics of SA immobilized beads Table 6 shows the physical and surface characteristics of SA immobilized beads. It was found that the surface charge of SA immobilized beads was lower than plain SA immobilized beads, but was much higher than the suspend biomass. Besides, the specific surface area and pore size on the surface of SA immobilized beads were higher than plain SA immobilized beads. The adsorption behavior of ammonium by SA immobilized beads can be explained by surface charge and area. The adsorption could take place due to the present of specific adsorption area, and the mutual attraction forces between ammonium and immobilized beads promoted the process of adsorption. In the FT-IR spectra of plain SA immobilized beads and SA immobilized beads, the strong band at 3426 cm1 was assigned to the stretching vibration of -OH functional group in SA. The characteristic bands between 1086 and 1033 cm1 which could be attributed to the deformation (or bending) of -OH functional group (Zhao et al., 2008). It is obvious that the band intensity at 3426 cm1, between 1086 and 1033 cm1 in SA immobilized beads were higher than plain SA immobilized beads. In addition, compared the spectrum of plain SA immobilized beads with that of SA immobilized beads, the bands at 1614 and 1423 cm1 were assigned to the presence of the antisymmetric and symmetric COO (Huang et al., 2000), which were shifted to the higher wavenumber of 1638 and 1429 cm1, respectively, and the intensity of both peaks attenuated. It was believed that all these phenomena in the intensity and band frequency were strongly affected by the internal or intramolecular hydrogen bonding of -OH groups. Since hydrogen bonds act as constraints to deformation vibrations, force constants for these vibrations are increased and wavenumber shifts to larger values (Huang et al., 2000). The peaks of SA immobilized beads at 1638 and 1429 cm1 shifted to large values due to
J. Yan, Y.Y. Hu / Bioresource Technology 100 (2009) 2341–2347 Table 6 Physical and surface characteristics of SA immobilized beads. Parameter Particle size (mm) Saturated wet density (g cm3) Zeta position (mV) BET surface area (m2 g1) Pore size (nm)
Suspended biomass
19.0
SA immobilized beads
Plain SA immobilized beads
3.6–3.8 1.286
3.2–3.5
43.8 0.5586
45.3 0.4810
1033.13
422.98
the formation of hydrogen bonding when biomass was added to the support. All the above differences demonstrated that SA immobilized beads was not the simply physical mixture of SA and biomass. The SA immobilized biomass beads were 3.7 mm in diameter on average. The surface of both SA immobilized biomass bead and plain SA immobilized bead showed some pores, which indicated the bead porosity of both beads. It was found that the surface of SA immobilized biomass bead was more porous and shaggy than that of plain SA immobilized bead, which confirmed the porosity of the bead. Each SA immobilized biomass bead was filled with cell mass. Some rod and arc shaped bacterium growed in the peripheral surface inner layer of the beads which was considered to be Nitrosomonas and Nitrovibrio, respectively. In addition, a porous structure was significantly observed in the inner space of SA immobilized biomass bead, such a formation was attributed to the diffusion of substrate and oxygen inside the bead. 3.5. Effect of HRT on partial nitrification An experiment was carried out to assess the effect of HRT on nitrification. The HRT of the nine reactors were set in the range of 2.5–35 h, as shown in Fig. 2. The concentration of ammonia decreased and the concentration of nitrite increased with HRT rising. COD removal rate increased rapidly with HRT rising when HRT was lower than 15 h, and when HRT was higher than 15 h, the reactors just showed a slightly higher COD removal rate. Remarkably, the nitrite production rate increased rapidly when COD removal rate was more than 80%, as well as the HRT was higher than 15 h. When organic carbon is present, heterotrophs compete with nitrifiers for oxygen, since the higher affinity of heterotrophs for oxygen (Philips et al., 2002), the degradation of COD took place fas-
Fig. 2. Evolution of concentration of ammonia and nitrite and COD removal rate at different HRT.
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ter than nitrification. At a shorter HRT, ammonia did not have a sufficient contact time to be converted to nitrite. It appears that for SA immobilized beads system, the reaction proceeds by two different stages, the nitrite production rate (NPR) was 0.0864 kg m3 d1 (R = 0.9977) in the lower HRT range (2.5–12.5 h). On the other hand, the NPR was up to 0.1364 kg m3 d1 (R = 0.9951) in the higher HRT range (15–35 h). The nitrite/ammonium ratio must be concerned for the possible subsequent Anammox process, the best nitrite/ammonium ratio for the Anammox process is about 1.3 (Feng et al., 2007). Based on the experimental data, the best HRT for subsequent Anammox process was 30 h, as the nitrite/ ammonium ratio was 1.42. 3.6. Effect of pH on partial nitrification Fig. 3 presents the results obtained for the pH effect experiments. The optimal partial nitrification took place at pH 8.0, and slight influence of pH was observed between pH 6.0 and 8.5, but at pH lower than 5.5 and higher than 9.0, inhibition of partial nitrification took place with nitrite accumulation decreased rapidly. The pH value in the system affects the nitrifying activity due to its effect on the NH3 =NHþ 4 -N and HNO2 =NO2 equilibrium. FA and FNA being inhibitory compounds for both ammonia and nitrite oxidizers and for nitrite oxidizers, respectively. Inhibition of AOB took place when FA and FNA are higher than 10 and 0.2 mg L1, respectively (Mosquera-Corral et al., 2005). As shown in Fig. 3, FA was lower than 10 mg L1 when pH was lower than 7.5 and FNA was lower than 0.2 mg L1 when pH was higher than 6.5, it indicated that the optimal partial nitrification in this case should take place when pH was between 6.5 and 7.5. Besides, the optimum activity of Nitrosomonas is reached at pH values between 7.9 and 8.2 (Alleman, 1984). Based on the discussion above, the optimal pH ranges for partial nitrification was broadened in SA immobilized beads system thanks to the ‘‘protective” effect of immobilization. 3.7. Effect of temperature on partial nitrification Nitrifying bacteria are known to be sensitive to temperature, and the effect of temperature on partial nitrification was investigated, as shown in Fig. 4. When the temperature was lower than 35 °C, both nitrite concentration and COD removal rate in the effluent were increased with temperature rising, as well as ammonia concentration decreased. But with more than 35 °C, ammonium concentration increased and nitrite concentration and COD removal rate decreased. The maximum NPR and COD removal rate were obtained (0.40 kg m3 d1 and 82.6%, respectively) when the temperature was 35 °C.
Fig. 3. Evolution of the concentration of nitrite, FA and FNA at different reactor pH value.
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Fig. 4. Evolution of the concentration of ammonia and nitrite and COD removal rate at different reactor temperature.
In order to understand the results obtained during this period, the NPR was calculated by Eq. (5) at different temperatures. The data fit a straight line and the parameters can be seen in Table 7. The activation energy obtained in this work actually lower than most of published data on ammonia oxidation with both free cells and immobilized cells, especially at the lower temperature range (15–20 °C), as shown in Table 7, which might increase the ammonia oxidation rate. It appears that for SA immobilized beads, thanks to the protective effect of immobilization (Morita et al., 2007; Isaka et al., 2007), the partial nitrification was accelerated at temperature between 15 °C and 35 °C compared with free cells system. Interestingly, many published research observed a breaking point at 20 °C and the nitrification reaction proceeds by two different regimes: one applying for the higher temperature (20– 30 °C or 20–35 °C) range and the other for the lower temperature range (10–20 °C), this phenomenon was not observed in this study. 3.8. Effect of DO on partial nitrification DO concentration is an utmost important parameter for both AOB and NOB. Fig. 5 showed the effect of DO concentration on ammonia consumption, nitrite and nitrate accumulation percentages in the reactors, as well as COD removal rate. Each experimental point was obtained in steady-state conditions after stabilizing DO concentration in the reactor. A region of maximum partial nitrification and COD removal in the DO concentration range of 4.2– 4.6 mg L1 existed with decreases in partial nitrification and COD removal at DO concentration ranges above or below those values. At DO concentration of 4.6 mg L1, nitrite accumulation rate and COD removal rate achieved maximum (63.1% and 66.3%, respec-
Table 7 Arrhenius parameters for partial nitrification. Temperature range (°C)
A Ea (kJ mol1)
15–35
42.1
10–35 10–20
73.5 162.7
20–35
55.8
10–20 20–30
87.1 38.6
R
Cell state
Reference
This study 2.97 106 0.999 SA immobilized 1.94 108 Free cells Benyahia and 2 1023 Polomarkaki (2005) 4 SA 1.74 10 immobilized 0.993 Free cells Kim et al. (2008) 0.992 Free cells
Fig. 5. Influence of DO on the percentage of ammonia consumption, nitrite accumulation and nitrate accumulation, as well as COD removal rate.
tively). Besides, nitrate accumulation was obviously observed when DO concentration was higher than 4.9 mg L1. The variations of the different substrates and products were similar to results obtained by other authors (Bernet et al., 2005; Ruiz et al., 2003; Cao et al., 2002) even though DO concentration was different at the maximum nitrite accumulation point. Ruiz et al. (2003) reported that the maximum was obtained at DO concentration of 0.7 mg L1 with complete ammonia consumption in free biomass reactor system. A maximum nitrite accumulation was observed by Bernet et al. (2005) with DO concentrations close to 2 mg/L in biofilm airlift reactor system. Cao et al. (2002) obtained the maximum nitrogen removal rate at DO concentration of 6 mg L1 in a PVA coimmobilized nitrifying and denitrifying bacteria. It can be seen that nitrite accumulation was limited by oxygen diffusion inside the biofilm or support. The bigger the oxygen transfer resistance was, the higher DO concentration was required to achieve the maximum partial nitrification. When DO was little, oxidization of ammonia was limited, however, when DO was too much, the activity of AOB decreased and the activity of NOB increased, so both nitrite accumulation and ammonia consumption weakened. 4. Conclusion SA was an excellent support for the immobilization of nitrifying biomass: the best HRT for subsequent Anammox process was 30 h; the optimal partial nitrification took place at the pH value of 8.0 and slight influence of pH was observed (from 6.0 to 8.5); the partial nitrification was accelerated at temperature between 15 °C and 35 °C compared with free cells system. A region of maximum partial nitrification and COD removal in the DO concentration range of 4.2–4.6 mg L1 existed. The effect of pH and temperature on partial nitrification was evidently weakened in SA immobilized biomass beads, but the effect of HRT and DO were reversely significant. Acknowledgements This research was supported by NSFC (Project No. 50678071) and GSFC (Project No. 06105409). The authors thank the WWTP operator for providing the inoculum. References Alleman, J.E., 1984. Elevated nitrite occurrence in biological wastewater treatment systems. Water Sci.Technol. 17, 409–419.
J. Yan, Y.Y. Hu / Bioresource Technology 100 (2009) 2341–2347 Ambrogi, V., Perioli, L., Maromttini, F., Accorsi, O., Pagano, C., Ricci, M., Rossi, C., 2008. Role of mesoporous silicates on carbamazepine dissolution rate enhancement. Micropor. Mesopor. Mat. 113, 445–452. American Public Health Association(APHA), 1995. Standard Methods for the Examination of Water and Wastewater. APHA, Washington. Benyahia, B., Polomarkaki, R., 2005. Mass transfer and kinetic studies under no cell growth conditions in nitrification using alginate gel immobilized Nitrosomonas. Process Biochem. 40, 1251–1262. Bernet, N., Sanchez, O., Cesbron, D., Steyer, J.P., Delgenès, J.P., 2005. Modeling and control of nitrite accumulation in a nitrifying biofilm reactor. Biochem. Eng. J. 24, 173–183. Cao, G.M., Zhao, Q.X., Sun, X.B., Zhang, T., 2002. Characterization of nitrifying and denitrifying bacteria coimmobilized in PVA and kinetics model of biological nitrogen removal by coimmobilized cells. Enzyme Microb. Technol. 30, 49– 55. Feng, Y.J., Tseng, S.K., Hsia, T.H., Ho, C.M., Chou, W.P., 2007. Partial nitrification of ammonium-rich wastewater as pretreatment for anaerobic ammonium oxidation (Anammox) using membrane aeration bioreactor. J. Biosci. Bioeng. 104, 182–187. Fierro, S., Sánchez-Saavedra, M.P., Copalcúa, C., 2008. Nitrate and phosphate removal by chitosan immobilized scenedesmus. Bioresour. Technol. 99, 1274– 1279. Fux, C., Boehler, M., Huber, P., Brunner, I., Siegrist, H., 2002. Biological treatment of ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium oxidation (Anammox) in a pilot plant. J. Biotechnol. 9, 295–306. Ganiué, R., López, H., Balaguer, M.D., 2007. Colprim J. Partial ammonium oxidation to nitrite of high ammonium content urban landfill leachates. Water Res. 41, 3317–3326. Hill, B.C., Khan, E., 2008. A comparative study of immobilized nitrifying and coimmobilized nitrifying and denitrifying bacteria for ammonia removal from sludge digester supernatant. Water Air Soil Poll. 195, 23–33. Huang, R.Y.M., Pal, R., Moon, G.Y., 2000. Pervaporation dehydration of aqueous ethanol and isopropanol mixtures through alginate/chitosan two ply composite membranes supported by poly (vinylidene fluoride) porous membrane. J. Membrane Sci. 167, 275–289. Isaka, K., Yoshie, S., Sumino, T., Inamori, Y., Tsuneda, S., 2007. Nitrification of landfill leachate using immobilized nitrifying bacteria at low temperatures. Biochem. Eng. J. 37, 49–55.
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Kim, S.K., Kong, I., Lee, B.H., Kang, L., Lee, M.G., Suh, K.H., 2000. Removal of ammonium-N from a recirculation aquacultural system using an immobilized nitrifier. Aquacult. Eng. 21, 139–150. Kim, J.H., Guo, X.J., Park, H.S., 2008. Comparison study of the effects of temperature and free ammonia concentration on nitrification and nitrite accumulation. Process Chem. 43, 154–160. Mallick, N., 2002. Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals 15, 377–390. Morita, M., Kudo, N., Uemoto, H., Watanabe, A., Shinozaki, H., 2007. Protective effect of immobilized ammonia oxidizers and phenol-degrading bacteria on nitrification in ammonia- and phenol containing wastewater. Eng. Life Sci. 7, 587–592. Mosquera-Corral, A., González, F., Campos, J.L., Méndez, R., 2005. Partial nitrification in a SHARON reactor in the presence of salts and organic carbon compounds. Process Biochem. 40, 3109–3118. Philips, S., Laanbroek, H.J., Verstraete, W., 2002. Origin, causes and effects of increased nitrite concentrations in aquatic environments. Environ. Sci. Technol. 1, 115–141. Rostron, W.M., Stuckey, D.C., Young, A.A., 2001. Nitrification of high strength ammonia wastewaters: comparative study of immobilisation media. Water Res. 35, 1169–1178. Ruiz, G., Jesion, D., Chamy, R., 2003. Nitrification with high nitrite accumulation for the treatment of wastewater with high ammonia concentration. Water Res. 37, 1371–1377. Seo, J.K., Jung, I.H., Kim, M.R., Kim, B.J., Nam, S.W., Kim, S.K., 2001. Nitrification performance of nitrifiers immobilized in PVA (polyvinyl alcohol) for a marine recirculation aquarium system. Aquacult. Eng. 24, 181–194. Shieh, W.K., Tsao, Y., 2004. Analysis of immobilized cell responses to substrate impulses. Biochem. Eng. J. 18, 177–184. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998. The Sequencing Batch Reactor as a powerful tool for the study of slowly growing anaerobic ammoniaoxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 596–598. Yamamoto, T., Takaki, K., Koyama, T., Kenji, Furukawa, 2008. Long-term stability of partial nitration of swine wastewater digester liquor and its subsequent treatment by Anammox. Bioresour. Technol. 99, 6419–6425. Zhao, K.Y., Cheng, G.X., Huang, J.J., Ying, X.G., 2008. Rebinding and recognition properties of protein-macromolecularly imprinted calcium phosphate/alginate hybrid polymer microspheres. React. Funct. Polym. 68, 732–741.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Partial nitrification to nitrite for treating ammonium-rich organic wastewater by immobilized biomass system J. Yan, Y.Y. Hu * School of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 23 July 2008 Received in revised form 19 November 2008 Accepted 20 November 2008 Available online 6 January 2009 Keywords: Ammonium-rich organic wastewater Partial nitrification Degradation of organics SA immobilized biomass beads Anammox
a b s t r a c t This study focused on the characteristics of the partial nitrification and degradation of organics with immobilized biomass beads in the treatment of ammonium-rich organic wastewater. Sodium alginate (SA) was selected as the best entrapment support after comparing partial nitrification rate and adsorption efficiency. The immobilization methods were optimized by an orthogonal experiment. Zeta position and BET surface area were used to explain the adsorption behavior of SA immobilized beads. FT-IR revealed that a SA immobilized biomass bead was not a simply physical mixture of SA and biomass. The porous structure of SA immobilized biomass beads were observed by scanning electron microscopy (SEM), which confirmed the porosity of the beads. According to the experimental data, the effects of pH and temperature on partial nitrification and COD removal were evidently weakened in SA immobilized biomass beads due to the ‘‘protective” effect of immobilization, whereas the effects of HRT and DO were enhanced. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Nitrogen has become a major problem in water quality management. Biological nitrification–denitrification is the most commonly used process for nitrogen removal from wastewater, especially municipal wastewater (Ruiz et al., 2003). However, these systems also have disadvantages, such as high operational costs of the processes because of the oxygen and organic carbon requirements. Compared to conventional nitrification and denitrification process, over 50% less oxygen demand and absence of organic carbon addition offer considerable cost savings in the combined partial nitrification/Anammox process (Fux et al., 2002). Some nitrogenous wastewater have high organic content. Since ammonia-oxidizing bacteria (AOB) might live with aerobic heterotrophic bacteria, partial nitrification could take place in combination with degradation of organic carbons (Yamamoto et al., 2008). Therefore, the combined partial nitrification/Anammox process might be a promising new method for the treatment of influents with high organic carbons and ammonium concentration. The growth rate of autotrophic AOB is lower than that of heterotrophic bacteria, with which they have to compete for oxygen, and without long retention times the suspended nitrifiers will be easily washed out of the reactor. The biomass concentration is increased by recirculation of the sludge after sedimentation, but limited by the efficiency of the sedimentation vessel. Besides, the ammonia oxidation rate is strongly influenced by the nature of nitrifying cul* Corresponding author. Tel.: +86 20 39380506; fax: +86 20 39380569. E-mail address: [email protected] (Y.Y. Hu). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.11.038
tures and a variety of environmental factors, including substrate concentration, dissolved oxygen (DO), temperature and pH. However, immobilization techniques can be used to overcome these problems. Immobilization is an efficient method to prevent biomass from being washed out and allow hyperconcentrated cultures. They can lead to relatively small reactors, provide some protection from adverse temperatures and toxic shocks, which would help in maintaining year-round treatment (Morita et al., 2007). Immobilized biomass can be divided into ‘‘naturally” attached biomass (biofilm) and ‘‘artificially” immobilized biomass. Biofilm has been widely applied in wastewater treatment. However, some particles can become anaerobic in the centre and settle to the reactor floor. One of the most common techniques for artificially immobilization is gel entrapment. Both natural and synthetic polymers can be used as the immobilization support, but it must fulfill various requirements, such as photo-transparency, non-toxicity, retention of cellular viability, and stability in the culture medium (Mallick, 2002). This immobilization technique is commonly used to immobilize a pure strain of bacteria because the mechanisms of pure strains are easy to understand (Fierro et al., 2008; Hill et al., 2008; Shieh and Tsao, 2004; Benyahia and Polomarkaki, 2005). Nevertheless, the immobilization of activated sludge has also been reported (Rostron et al., 2001; Seo et al., 2001; Isaka et al., 2007). Compared to pure strain of bacteria, immobilization of activated sludge could remove multiple pollutants due to the biodiversity of the activated sludge. This study focuses on the characteristics of the partial nitrification and degradation of organics with an immobilized biomass in
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treating ammonium-rich organic wastewater. It serves as a first step in the Anammox process with partial denitrification via nitrite. After four materials, i.e. sodium carboxymethylcellulose (Na-CMC), sodium alginate (SA), polyvinyl alcohol and sodium alginate (PVA–SA), and chitosan (CTS), were prepared as supports for entrapping the biomass, the best entrapment support were selected by comparing partial nitrification rate and adsorption efficiency. Then, the best immobilization method was obtained by orthogonal experiment and the physicochemical characteristics of immobilized beads were investigated. Finally, the hydraulic retention time (HRT), pH, temperature and dissolved oxygen (DO) tolerance for immobilized beads were studied. The objective of this work was to determine if immobilization has advantages in reactor operation when pH, DO and temperature were variable under ammonia and COD loading applied. 2. Methods 2.1. Immobilization methods 2.1.1. Reagents The immobilization supports, Na-CMC (high viscosity; 2.0% in H2O at 25 °C: 300–600 mPa s; content of sodium: 6.5–6.8%), were supplied by Fuchen Chemical Enterprise Co. in Tianjin, China. Sodium alginate (SA; viscosity: 150 mPa s) was chemically pure grade. Polyvinyl alcohol (PVA) was analytically pure grade, and CTS (degree of deacetylation: 75%) was obtained from Boao Biotechnology Ltd. (Shanghai, China). 2.1.2. Acclimation of biomass Biomass were obtained from the activated sludge of the aeration tank of an urban wastewater treatment plant (WWTP) in Guangzhou, China, and acclimated with synthetic ammonia and COD feeding for two month in two 5 L sequencing batch reactor (SBR) and a volume exchange ratio of about 0.5. Both reactors were provided with a thermostatic jacket and temperature were maintained at 30 ± 1 °C using thermostatic bath. Both reactors were mechanically stirrered (JB300-D, Biaoben, Chia). Two pumps (Beta/4a 0708, Prominent, Germany) and oxygen valves were necessary to operate the reactor. The influent was maintained at 4 °C in a 10 L tank. Both reactors were drawn by gravity discharge using an electro-valve. Dissolved oxygen (DO) was measured with a electrode (550A, YSI, USA) and controlled at 0.5 mg L1 by adjusting the air flow rate manually. The pH was measured with a electrode (pH 6, Ecoscan, Singapore) and adjusted to about 7.5 by addition of acid (H2SO4 1 M) or alkali (NaOH 1 M) solution, respectively. The composition of the feeding solution was shown in Table 1, the trace solution was according to Strous et al. (1998). Ammonia, nitrite, nitrate and COD concentrations were measured everyday. 2.1.3. Preparation of immobilization beads The preparation method of immobilized beads was described as follow. The acclimated biomass was washed by distilled water three times before use. The biomass was concentrated by centrifuge at 1000 rpm for 5 min. One portion of the concentrated
Table 1 Composition of feeding solution. Parameter
g L1
Parameter
g L1
NH4Cl CaCl2 KH2PO4 MgSO4 FeSO4 7H2O
1.383 0.30 0.07 0.02 0.009
NaHCO3 EDTA Traces solution C2H12O6
2.16 0.006 1.25 mL L1 0.156
biomass (28.8 g VSSL1) was mixed thoroughly with one portion of the support solution as shown in Table 2. This mixture was then pumped (7524-55, Cole-Parmer Instrument, USA) into kieselgel tube with pinhead for drop formation (Pure support solution was pumped into kieselgel tube for preparing plain immobilized beads) (Seo et al., 2001). The extruded drops fell into a crosslink solution to form solid beads. The beads were transferred to a stabilization solution with aeration at 30 °C, the stabilization solution was prepared by mixing the cross linking agent with the feeding solution as shown in Table 1, and the pH of the stabilization solution was
Table 2 Immobilization condition for four kinds of beads. Support aqueous solution (w/v)
Crosslink solution (w/v)
Crosslink time (h)
SA (5%) PVA (10%)-SA (2%) Na-CMC (2%) CTS (3%)a
4% CaCl2 4% CaCl2 21.6% FeCl3 1% Polyphosphoric acid (PPA)
12 12 1 6
a The chitosan solution was prepared by mixing chitosan flakes with 5% acetic acid.
Table 3 Operation condition for each reactor at different periods. Study at constant DO = 4 mg L1, pH 7.5, T = 30 °C
Study at constant pH 7.5, T = 30 °C
Period A-support study
Period B-support absorption efficiency study
No.
Support
No.
Support
1 2 3
SA bead CTS bead CMC bead
1 2 3
Plain SA bead Plain CTS bead Plain CMC bead Plain PVA–SA bead
4
PVA–SA bead Study at constant T = 30 °C, DO = 4 mg L1, pH 7.5 Period D-HRT study
4 Study at constant T = 30 °C, DO = 4 mg L1, HRT = 30 h Period E-pH study
No.
HRT (h)
No.
pH
1 2 3 4 5 6 7 8 9
2.5 5.0 7.5 10.0 12.5 15.5 20.0 30.0 35.0
1 2 3 4 5 6 7 8 9 10
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
Study at constant DO = 4 mg L1, pH 8, HRT = 30 h
Study at constant T = 30 °C, pH 8, HRT = 30 h
Period F-temperature study
Period G-DO study
No.
T (°C)
No.
DO (mg L1)
1 2 3 4 5 6
15 20 25 30 35 40
1 2 3 4 5 6 7 8 9
1.8 2.1 3.2 3.4 3.9 4.2 4.6 5.1 5.2
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adjusted to about 7.5. At last, removed and rinsed the beads with distilled water three times. Additional activity recovery time was not necessary in this method (Kim et al., 2000).
Electrophoretic mobilities of particles were measured by the instrument and converted to zeta potential by using Smoluchowski equation.
2.2. Experimental method and procedure
2.4. Surface area and porosity
The batch experiments were carried out in 10 SBRs of 1 L and a volume exchange ratio of about 0.8. A 200 mL of immobilized beads was added to each reactor (20% reactor fill). All the reactors were magnetic stirred. A PVC net was installed at the bottom of the reactor (pore size 1 mm) to keep the immobilized beads away from the rotor. Other parts of the experimental system were operated as described above (Section 2.1.2.) The experimental time covered 7 periods. The performance of each reactor was different in each period. A summary of the operating conditions for each reactor is given in Table 3. The composition of the feeding solution shown in Table 1 is during the experiment except period B and C. During period B, it was done with one litre of feed with NHþ 4 -N concentration of 1 . Period C 1000 mg L1 and NO 2 -N concentration of 550 mg L was the orthogonal experiment for optimizing the immobilization method, and the orthogonal experiment with three levels and four factors were designed. The four factors were the concentration of support solution, mixing volume ratio of support solution and concentrated biomass, the concentration of cross linking agent and cross linking time. The three levels of each factor are showed in Table 4. The arrangement of orthogonal experiment was listed in Table 5.
Adsorption–desorption isotherms 77 K of both immobilized biomass and plain beads were determined using a computer controlled micromeritics ASAP 2020M (USA) apparatus. The specific surface area and pore size were determined by application of the Brunauer, Emmett and Teller (BET) method to the adsorption data (Ambrogi et al., 2008).
2.3. Zeta position A sample of 2 g immobilized SA bead was added to 100 mL deionized water and then mixed at 1500 rpm for 24 h by magnetic stirrer (ML-902, Shanghai suhao Instrument Ltd., China) kept in biochemical incubator (SPX-150B-Z, Shanghai China) at 30 ± 0.5 °C to obtain the suspension. Zeta potential measurements of the suspension obtained were carried out by Nano-ZS model (3800, Malvern Instrument Ltd., UK) zetasizer instrument. Table 4 Factors and levels of orthogonal experiment. No.
SA% (w/v)
SA:concentrated biomass (v/v)
CaCl2 (w/v)
Cross linking time (h)
1 2 3
5 7 10
1:1 1:1.5 1:2
2 4 8
12 24 48
Table 5 Analysis of crosstab data.
2.5. Infrared spectroscopy FT-IR spectroscopy was conducted on the immobilized biomass and plain beads. The immobilized beads were fully washed with deionized water, dried and ground to powder. Then, the powder was mixed with KBr and analyzed by an FT-IR spectrometer (Vector 33, Bruker Instrument Ltd., Germany). The experiments were run with air as the background, and the resolution and number of scans were 4.0 cm1 and 32, respectively. 2.6. Scanning electron microscopy Scanning electron microscopy (SEM) was used to study the cross-section morphology of the immobilized beads and to compare the surface characteristic among free biomass, immobilized biomass beads and plain beads. A ESEM (XL-30, Philips Instrument Ltd., Netherlands) was used for the specimens at 10 kV. 2.7. Analytical methods Volatile suspended solids (VSS), chemical oxygen demand (COD), ammonium ðNHþ 4 -NÞ, nitrites ðNO2 -NÞ and nitrates ðNO3 -NÞ were measured according to standard methods (APHA, 1995). The volume of immobilized bead was measured by draining method, which measured the volume of mixed liquor displaced when immobilized beads were added. The average wet density of immobilized bead was calculated by the average wet quality and volume. Concentrations of free ammonia (FA) and free nitrous acid (FNA) were calculated as a function of pH, temperature and total ammonium as nitrogen (TAN), for FA, or total nitrite (TNO2), for FNA (Ganiué et al., 2007):
FA ðmg NL1 Þ ¼
No.
SA% (w/v)
SA:concentrated biomass (v/v)
CaCl2 (w/v)
Cross linking time (h)
Concentration nitrite (mg L1)
1 2 3 4 5 6 7 8 9 k1 k2 k3 Rang
5% 7% 10% 5% 7% 10% 5% 7% 10% 202.26 191.22 191.00 11.26
1:1 1:1 1:1 1:1.5 1:1.5 1:1.5 1:2 1:2 1:2 140.44 195.12 248.92 108.48
4% 2% 8% 2% 8% 4% 8% 4% 2% 197.00 168.78 220.7 53.92
48 12 24 24 48 12 12 24 48 225.74 217.02 141.72 84.02
22.24 56.64 61.56 75.64 54.76 64.72 104.38 79.82 64.72
Note: Rang = kmaxkmin, k: sum of each level, concentrations of nitrite were average values.
TAN
1 þ ð10pH =K NH e Þ TNO2 1 FNA ðmg NL Þ ¼ pH 1 þ ðK NO Þ e =10
ð1Þ ð2Þ
¼ e6344=ð273þTÞ K NH e
ð3Þ
K NO e
ð4Þ
2300=ð273þTÞ
¼e
The activation energy of a reaction can be determined graphically by taking the natural logarithm of Arrhenius equation which describes the relationship between the reaction rate and the temperature, as shown in the following equation. Where A is the frequency factor for the reaction, R is the universal gas constant, T is the temperature (K), and Ea is the activation energy.
ln K ¼
Ea þ ln A RT
ð5Þ
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3. Results and discussion
3.3. Optimizing the SA immobilization method
3.1. Partial nitrification by different supporting immobilized beads
Based on the discussion above, SA might be an excellent support for partial nitrification. In order to obtain the optimal immobilization method, an orthogonal experiment was carried out. The arrangements of orthogonal experiments were listed in Table 5, which including nine sets of experiments, the result was the concentration of nitrite when HRT was 30 h. The last four rows of Table 5 show the results of different factors affecting on the partial nitrification processes by SA immobilized beads. The most significant effect among the four factors on partial nitrification was found to be the mixing volume ratio of support solution and concentrated biomass, followed by the concentration of cross linking agent, cross linking time and the concentration of support solution. The change of the concentration of support solution did not show significant effect on the partial nitrification, so the concentration of support solution of 5% was recommended for the immobilization process. The concentration of nitrite in the effluent of the reactor increased conspicuously with the increase of the mixing volume ratio of support solution and concentrated biomass, it means that the optimal mixing ratio could be adjusted to 1:2 in order to achieve a higher nitrite accumulation rate. A further increase of the concentration of cross linking agent may also improve partial nitrification efficiency. On the contrary, concentration of nitrite in the effluent of the reactor decreased significantly with the increase in cross linking time, so the cross linking time of 12 h was recommended. From the study mentioned above, the optimum immobilization methods were determined, which were: mixing volume ratio of support solution concentrated biomass of 1:2, concentration of cross linking agent of 8% , cross linking time of 12 h and concentration of support solution of 5%.
Nitrite production by SA immobilized bead was the highest, the concentration of nitrite was 28.85 mg L1 and 105.71 mg L1 when HRT was 15 h and 30 h, respectively. CMC immobilized bead showed the lowest partial nitrification efficiency, the concentration of nitrite was 0.25 mg L1 and 2.40 mg L1 when HRT was 15 h and 30 h, respectively. The order of partial nitrification efficiencies were SA > PVA–SA > CTS > CMC. (For PVA–SA immobilized bead, the concentration of nitrite was 22.04 mg L1 and 76.73 mg L1 when HRT was 15 h and 30 h, respectively. For CTS immobilized bead, the concentration of nitrite was 1.5 mg L1 and 29.9 mg L1 when HRT was 15 h and 30 h, respectively.) No nitrate was detected during the whole experiment. There were similar phenomena observed in nitrification systems by four immobilized beads that the sum of the dissolved nitrogen compounds in the effluent was lower compared with the inlet ammonium concentration. The most serious nitrogen loss occurred (about 84.2%) in CMC immobilized bead system. It suggested that adsorption of nitrogen by immobilized beads might occur in these systems. 3.2. Adsorption behavior of plain gel beads It was found that all the four plain gel beads could adsorb ammonia, as shown in Fig. 1. SA plain gel beads had 10.9% and 12.6% removal in 22 h and 40 h, respectively. Adsorption efficiency by CTS plain gel beads was also relatively high (9.4% and 9.5% in 22 h and 40 h, respectively). The order of adsorption of ammonium was SA > CTS > CMC > PVA. Fig. 1 also showed the adsorption of nitrite by four plain gel beads. CTS plain gel beads had significant 13.3% and 28.0% removal in 22 h and 40 h, respectively. Adsorption efficiency by CMC plain gel beads was also relatively high (13.9% and 22.1% in 22 h and 40 h, respectively). This result could explain the extraordinary low nitrite accumulation in CTS and CMC immobilized beads system. The order of adsorption of nitrite was CTS > CMC > SA > PVA. Since nitrite is the oxidation production of ammonia-oxidizing bacteria (AOB), the aim of partial nitrification is to obtain the nitrite accumulation. Thus, the adsorption of nitrite should be minimized in order to obtain the nitrite accumulation, and the adsorption of ammonia was less important compared with nitrite.
Fig. 1. Adsorption efficiency of ammonium and nitrate by plain gel beads with time.
3.4. Characteristics of SA immobilized beads Table 6 shows the physical and surface characteristics of SA immobilized beads. It was found that the surface charge of SA immobilized beads was lower than plain SA immobilized beads, but was much higher than the suspend biomass. Besides, the specific surface area and pore size on the surface of SA immobilized beads were higher than plain SA immobilized beads. The adsorption behavior of ammonium by SA immobilized beads can be explained by surface charge and area. The adsorption could take place due to the present of specific adsorption area, and the mutual attraction forces between ammonium and immobilized beads promoted the process of adsorption. In the FT-IR spectra of plain SA immobilized beads and SA immobilized beads, the strong band at 3426 cm1 was assigned to the stretching vibration of -OH functional group in SA. The characteristic bands between 1086 and 1033 cm1 which could be attributed to the deformation (or bending) of -OH functional group (Zhao et al., 2008). It is obvious that the band intensity at 3426 cm1, between 1086 and 1033 cm1 in SA immobilized beads were higher than plain SA immobilized beads. In addition, compared the spectrum of plain SA immobilized beads with that of SA immobilized beads, the bands at 1614 and 1423 cm1 were assigned to the presence of the antisymmetric and symmetric COO (Huang et al., 2000), which were shifted to the higher wavenumber of 1638 and 1429 cm1, respectively, and the intensity of both peaks attenuated. It was believed that all these phenomena in the intensity and band frequency were strongly affected by the internal or intramolecular hydrogen bonding of -OH groups. Since hydrogen bonds act as constraints to deformation vibrations, force constants for these vibrations are increased and wavenumber shifts to larger values (Huang et al., 2000). The peaks of SA immobilized beads at 1638 and 1429 cm1 shifted to large values due to
J. Yan, Y.Y. Hu / Bioresource Technology 100 (2009) 2341–2347 Table 6 Physical and surface characteristics of SA immobilized beads. Parameter Particle size (mm) Saturated wet density (g cm3) Zeta position (mV) BET surface area (m2 g1) Pore size (nm)
Suspended biomass
19.0
SA immobilized beads
Plain SA immobilized beads
3.6–3.8 1.286
3.2–3.5
43.8 0.5586
45.3 0.4810
1033.13
422.98
the formation of hydrogen bonding when biomass was added to the support. All the above differences demonstrated that SA immobilized beads was not the simply physical mixture of SA and biomass. The SA immobilized biomass beads were 3.7 mm in diameter on average. The surface of both SA immobilized biomass bead and plain SA immobilized bead showed some pores, which indicated the bead porosity of both beads. It was found that the surface of SA immobilized biomass bead was more porous and shaggy than that of plain SA immobilized bead, which confirmed the porosity of the bead. Each SA immobilized biomass bead was filled with cell mass. Some rod and arc shaped bacterium growed in the peripheral surface inner layer of the beads which was considered to be Nitrosomonas and Nitrovibrio, respectively. In addition, a porous structure was significantly observed in the inner space of SA immobilized biomass bead, such a formation was attributed to the diffusion of substrate and oxygen inside the bead. 3.5. Effect of HRT on partial nitrification An experiment was carried out to assess the effect of HRT on nitrification. The HRT of the nine reactors were set in the range of 2.5–35 h, as shown in Fig. 2. The concentration of ammonia decreased and the concentration of nitrite increased with HRT rising. COD removal rate increased rapidly with HRT rising when HRT was lower than 15 h, and when HRT was higher than 15 h, the reactors just showed a slightly higher COD removal rate. Remarkably, the nitrite production rate increased rapidly when COD removal rate was more than 80%, as well as the HRT was higher than 15 h. When organic carbon is present, heterotrophs compete with nitrifiers for oxygen, since the higher affinity of heterotrophs for oxygen (Philips et al., 2002), the degradation of COD took place fas-
Fig. 2. Evolution of concentration of ammonia and nitrite and COD removal rate at different HRT.
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ter than nitrification. At a shorter HRT, ammonia did not have a sufficient contact time to be converted to nitrite. It appears that for SA immobilized beads system, the reaction proceeds by two different stages, the nitrite production rate (NPR) was 0.0864 kg m3 d1 (R = 0.9977) in the lower HRT range (2.5–12.5 h). On the other hand, the NPR was up to 0.1364 kg m3 d1 (R = 0.9951) in the higher HRT range (15–35 h). The nitrite/ammonium ratio must be concerned for the possible subsequent Anammox process, the best nitrite/ammonium ratio for the Anammox process is about 1.3 (Feng et al., 2007). Based on the experimental data, the best HRT for subsequent Anammox process was 30 h, as the nitrite/ ammonium ratio was 1.42. 3.6. Effect of pH on partial nitrification Fig. 3 presents the results obtained for the pH effect experiments. The optimal partial nitrification took place at pH 8.0, and slight influence of pH was observed between pH 6.0 and 8.5, but at pH lower than 5.5 and higher than 9.0, inhibition of partial nitrification took place with nitrite accumulation decreased rapidly. The pH value in the system affects the nitrifying activity due to its effect on the NH3 =NHþ 4 -N and HNO2 =NO2 equilibrium. FA and FNA being inhibitory compounds for both ammonia and nitrite oxidizers and for nitrite oxidizers, respectively. Inhibition of AOB took place when FA and FNA are higher than 10 and 0.2 mg L1, respectively (Mosquera-Corral et al., 2005). As shown in Fig. 3, FA was lower than 10 mg L1 when pH was lower than 7.5 and FNA was lower than 0.2 mg L1 when pH was higher than 6.5, it indicated that the optimal partial nitrification in this case should take place when pH was between 6.5 and 7.5. Besides, the optimum activity of Nitrosomonas is reached at pH values between 7.9 and 8.2 (Alleman, 1984). Based on the discussion above, the optimal pH ranges for partial nitrification was broadened in SA immobilized beads system thanks to the ‘‘protective” effect of immobilization. 3.7. Effect of temperature on partial nitrification Nitrifying bacteria are known to be sensitive to temperature, and the effect of temperature on partial nitrification was investigated, as shown in Fig. 4. When the temperature was lower than 35 °C, both nitrite concentration and COD removal rate in the effluent were increased with temperature rising, as well as ammonia concentration decreased. But with more than 35 °C, ammonium concentration increased and nitrite concentration and COD removal rate decreased. The maximum NPR and COD removal rate were obtained (0.40 kg m3 d1 and 82.6%, respectively) when the temperature was 35 °C.
Fig. 3. Evolution of the concentration of nitrite, FA and FNA at different reactor pH value.
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J. Yan, Y.Y. Hu / Bioresource Technology 100 (2009) 2341–2347
Fig. 4. Evolution of the concentration of ammonia and nitrite and COD removal rate at different reactor temperature.
In order to understand the results obtained during this period, the NPR was calculated by Eq. (5) at different temperatures. The data fit a straight line and the parameters can be seen in Table 7. The activation energy obtained in this work actually lower than most of published data on ammonia oxidation with both free cells and immobilized cells, especially at the lower temperature range (15–20 °C), as shown in Table 7, which might increase the ammonia oxidation rate. It appears that for SA immobilized beads, thanks to the protective effect of immobilization (Morita et al., 2007; Isaka et al., 2007), the partial nitrification was accelerated at temperature between 15 °C and 35 °C compared with free cells system. Interestingly, many published research observed a breaking point at 20 °C and the nitrification reaction proceeds by two different regimes: one applying for the higher temperature (20– 30 °C or 20–35 °C) range and the other for the lower temperature range (10–20 °C), this phenomenon was not observed in this study. 3.8. Effect of DO on partial nitrification DO concentration is an utmost important parameter for both AOB and NOB. Fig. 5 showed the effect of DO concentration on ammonia consumption, nitrite and nitrate accumulation percentages in the reactors, as well as COD removal rate. Each experimental point was obtained in steady-state conditions after stabilizing DO concentration in the reactor. A region of maximum partial nitrification and COD removal in the DO concentration range of 4.2– 4.6 mg L1 existed with decreases in partial nitrification and COD removal at DO concentration ranges above or below those values. At DO concentration of 4.6 mg L1, nitrite accumulation rate and COD removal rate achieved maximum (63.1% and 66.3%, respec-
Table 7 Arrhenius parameters for partial nitrification. Temperature range (°C)
A Ea (kJ mol1)
15–35
42.1
10–35 10–20
73.5 162.7
20–35
55.8
10–20 20–30
87.1 38.6
R
Cell state
Reference
This study 2.97 106 0.999 SA immobilized 1.94 108 Free cells Benyahia and 2 1023 Polomarkaki (2005) 4 SA 1.74 10 immobilized 0.993 Free cells Kim et al. (2008) 0.992 Free cells
Fig. 5. Influence of DO on the percentage of ammonia consumption, nitrite accumulation and nitrate accumulation, as well as COD removal rate.
tively). Besides, nitrate accumulation was obviously observed when DO concentration was higher than 4.9 mg L1. The variations of the different substrates and products were similar to results obtained by other authors (Bernet et al., 2005; Ruiz et al., 2003; Cao et al., 2002) even though DO concentration was different at the maximum nitrite accumulation point. Ruiz et al. (2003) reported that the maximum was obtained at DO concentration of 0.7 mg L1 with complete ammonia consumption in free biomass reactor system. A maximum nitrite accumulation was observed by Bernet et al. (2005) with DO concentrations close to 2 mg/L in biofilm airlift reactor system. Cao et al. (2002) obtained the maximum nitrogen removal rate at DO concentration of 6 mg L1 in a PVA coimmobilized nitrifying and denitrifying bacteria. It can be seen that nitrite accumulation was limited by oxygen diffusion inside the biofilm or support. The bigger the oxygen transfer resistance was, the higher DO concentration was required to achieve the maximum partial nitrification. When DO was little, oxidization of ammonia was limited, however, when DO was too much, the activity of AOB decreased and the activity of NOB increased, so both nitrite accumulation and ammonia consumption weakened. 4. Conclusion SA was an excellent support for the immobilization of nitrifying biomass: the best HRT for subsequent Anammox process was 30 h; the optimal partial nitrification took place at the pH value of 8.0 and slight influence of pH was observed (from 6.0 to 8.5); the partial nitrification was accelerated at temperature between 15 °C and 35 °C compared with free cells system. A region of maximum partial nitrification and COD removal in the DO concentration range of 4.2–4.6 mg L1 existed. The effect of pH and temperature on partial nitrification was evidently weakened in SA immobilized biomass beads, but the effect of HRT and DO were reversely significant. Acknowledgements This research was supported by NSFC (Project No. 50678071) and GSFC (Project No. 06105409). The authors thank the WWTP operator for providing the inoculum. References Alleman, J.E., 1984. Elevated nitrite occurrence in biological wastewater treatment systems. Water Sci.Technol. 17, 409–419.
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