nutrient removal performance in an intermittent

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Added CH3COONa to reach final conc. of 600 mg phosphate uptake. COD/l, then aeration after 3 hours of phosphate release. PNO2. End of 2nd aerobic stage.
Journal of the Chinese Institute of Engineers, Vol. 33, No. 4, pp. 581-590 (2010)

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NUTRIENT REMOVAL PERFORMANCE IN AN INTERMITTENT AERATED SEQUENCING BATCH MEMBRANE BIOREACTOR

Wen-Shang Chou*, Tien-Chin Chang, Sheng-Jie You, and Yao-Chon Sie

ABSTRACT In this study, a sequencing batch membrane bioreactor (SBMBR) was operated with intermittent aeration to perform partial nitrification nutrient removal. Several batch experiments were also carried out to investigate the nutrient removal characteristics. The removal efficiencies of suspended solid (SS), chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) for the SBMBR process were 96.7, 95.5, 73.6 and 98.1%, respectively. It was also found that all the organic- and ammonia-nitrogen was nitrified into nitrite rather than nitrate form, showing partial nitrification. The results of the batch experiments showed that heterotrophic nitrification occurred and nitrite was still the predominant oxidized nitrogen species. It was also observed that autotrophic denitrification occurred during the denitrification batch experiments, although the autotrophic denitrification rate was only 34% of the heterotrophic denitrification rate. In addition, there was no significant difference in the phosphate uptake rate observed between nitrite and nitrate as electron acceptors. Finally, five nutrient removal related bacteria were identified in the SBMBR reactor using the pure culture method. Key Words: nitrogen, phosphorus, membrane, cloning, activated sludge.

I. INTRODUCTION Typically the biological nutrient removal (BNR) process is configured with anaerobic, anoxic and aerobic treatment zones for simultaneous carbon, nitrogen and phosphorus removal (Metcalf and Eddy, 2003). Anoxic and aerobic tanks are needed for nitrogen removal. During nitrification, ammonia-nitrogen is nitrified into nitrite- or nitrate-nitrogen in the aerobic tank. The nitrifying liquid is recycled into the anoxic tank where denitrification is performed. On the other hand, the anaerobic-aerobic zones are designed to enhance the growth of phosphate accumulating organisms (PAO) *Corresponding author. (Tel: 886-2-2917-3282; Fax: 886-22918-0335; Email: [email protected]) W.-S. Chou and T.-C. Chang are with Institute of Environmental Planning and Management, National Taipei University of Technology, Taipei 106, Taiwan, R.O.C. S.-J. You is with Department of Bioenvironmental Engineering and R&D Center for Membrane Technology, Chung Yuan Christian University, Chungli 320, Taiwan, R.O.C. Y.-C. Sie is with Department of Civil Engineering, Chung Yuan Christian University, Chungli 320, Taiwan, R.O.C.

for phosphorus removal. The PAO obtain energy by degrading intracellular polyphosphates for the uptake of the soluble organic substrate which is subsequently stored as intracellular polyhydroxyalkanoates (PHA) under anaerobic conditions. In the following aerobic conditions, the PHA is consumed for excess phosphateuptake and growth. However, in the BNR process there is competition between the denitrifying bacteria and the PAO for carbon. This problem can be solved by the utilization of some kinds of PAO, namely denitrifying phosphate accumulating organisms (DNPAO) (Kerrn-Jespersen and Henze, 1993) which can take up phosphates under both anoxic and aerobic conditions. These DNPAO can utilize nitrates and nitrites as electron acceptors for simultaneous denitrification and phosphate-uptake with very little carbon consumption. There have been efforts made in several studies to enhance the DNPAO in the BNR processes by enlarging the anoxic tank and decreasing the size of the aerobic tank (Kuba et al., 1994; Meinhold et al., 1999; You et al., 2005). However, decreasing the size of the aerobic tank can lead to a decrease in the nitrifying

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Journal of the Chinese Institute of Engineers, Vol. 33, No. 4 (2010)

reaction time resulting in low nitrification performance. Thus, although the anoxic phosphate uptake has been enhanced, the overall nitrogen removal performance may be poorer. Fortunately, this problem can be solved by using membrane bioreactors (MBR), which have started operating worldwide in municipal and industrial wastewater treatment. The advantages of the MBR process are the high quality of the effluent and the complete removal of most microorganisms, while the disadvantages are aeration limitations, membrane fouling, and membrane costs (Stephenson et al., 2000). In the MBR process, the membrane replaces the sedimentation tank of the traditional activated sludge process, including the BNR process. In past studies the MBR process has most often operated with a moderate to high solid retention time (SRT) in order to increase the high biomass concentration, high loading rate capability, and high permeate flux. This means that some nitrifying bacteria, including ammonia oxidizing bacteria (AOB) or nitrite oxidizing bacteria (NOB), are enriched to enhance the nitrification performance in the MBR reactor. In addition, since most of the bacteria are retained in the membrane, the operating parameters of the traditional activated sludge process can be modified without raising concerns about the bulking problem. For example, even under high biomass and low dissolved oxygen (DO) conditions the MBR offers partial nitrification performance, i.e., ammonia is oxidized into nitrite but not further into nitrate. The first step in the nitrification process is the oxidization of ammonia nitrogen to form nitrite by AOB, followed by the oxidization of nitrite to form nitrate by NOB. Both AOB and NOB need oxygen for the oxidization process, while the nitrification process needs energy for aeration of the activated sludge. The accumulation of nitrite (as opposed to nitrate) in the aerobic tank could lead to lower aeration energy cost, meaning the size of the aerobic tank also could be decreased. In addition, since the denitrifier needs carbon for denitrification, a nitrite accumulation process would also decrease the carbon requirement for denitrification, compared with a nitrate accumulating process (Metcalf and Eddy, 2003). Partial nitrification has several advantages, such as it needs lower oxygen consumption for nitrification and lower amounts of organic materials for denitrification and also lower sludge production for nitrification. Several researchers have studied the performance of partial nitrification membrane bioreactors based on low dissolved oxygen control or intermittent aeration operation (Terada et al., 2004; Wyffels et al., 2004a, b; Yoo et al., 2006). Given suitable operating conditions, it will be very easy to produce a nitrite accumulation bioreactor due to the high biomass concentration in the MBR process.

However, only very few have studied the microbial diversity in these partial nitrification MBR processes (Wyffels et al., 2004b; Yoo et al., 2006). In this study, a nitrite accumulating intermittent aeration sequencing batch membrane bioreactor (SBMBR) was operated to enhance partial nitrification performance. Several batch experiments were designed for evaluating nitrogen and phosphorus removal efficiency. Finally, the traditional culturing method was used to identify the microbial diversity of the SBMBR process. II. MATERIALS AND METHODS 1. Process and Operation Conditions Figure 1(a) shows the configuration of the SBMBR for intermittent aeration operation at room temperature. Two hollow-fiber membranes (Sterapore HF, Mitsubishi Rayon Co. Ltd, Japan), with a total area of 0.4 m2, were installed in a 28 liter activated sludge tank. The material of the membranes was polyethylene with a separation particle size of 0.4 µm. The MBRs were drained out by a peristaltic pump. The pump was powered for 12 minutes for permeation drainage, followed by 3 minutes of power off to allow the membrane to relax (according to the manufacturer’s suggestions). The SBMBR reactor was operated with an anaerobic-aerobic-anoxic-aerobic sequence, as shown in Fig. 1(b). The sludge retention time was 30 days with a hydraulic retention time of 48 hours. The MLSS (mixed liquid suspended solid, most often referred to as biomass) was maintained in a steady state at 8,300 mg/l, with a food to microorganism ratio (F/M ratio) of 0.1 kg . BOD/kg . MLSS . d. The pH ranged from 6.8 to 7.2 and the dissolved oxygen (DO) in the aerobic phase was less than 1.0 mg/l, probably due to the high MLSS concentration. The substrate used for the reactor cultivation contained (per liter): milk powder, 163.2 mg; sucrose, 16.2 mg; acetate, 37.6 mg; (NH4) 2SO 4, 78 mg; urea, 30 mg; FeCl3, 0.1 mg; and KH2PO4, 15 mg. The concentrations of COD, TKN (organic plus ammonia nitrogen), and TP in the wastewater were 300 mg/l, 40 mg-N/l and 5 mg-P/l, respectively, simulating settled wastewater from the Min-Sheng Municipal Wastewater Treatment Plant, Taipei. 2. Batch Experiments (i) Batch Experiments Showing Aerobic Nitrification and Anoxic Denitrification Performance In this study, a series of batch aerobic nitrification experiments were carried out in order to confirm the autotrophic- and heterotrophic-nitrification performance. Results are shown in Table 1. In the

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W. S. Chou et al.: Performance of a Sequential Batch Membrane Bio-Reactor for Nutrient Removal

Table 1 Batch experiment design Objective of the experiments

No.

Aerobic nitrification

AN-1 HN-1

End of anaerobic stage End of anaerobic stage

AN-2 AN-3

End of anoxic stage End of anoxic stage, diluted 4 times.

ADN-1 HDN-1

End of 2 nd aerobic stage End of 2 nd aerobic stage

HDN-2

End of 1 st aerobic stage

ADN-2 HDN-3

End of 1 st aerobic stage End of 2 nd aerobic stage

HDN-4

End of 1 st aerobic stage

Anoxic denitrification

Aerobic/Anoxic P O2 phosphate uptake

Activated source

Substrate additions

Added KNO 2 to reach final conc. of 50 mg N/l. Added KNO2 to reach final conc. of 50 mg N/l, and CH 3COONa to reach final conc. of 300 mg COD/l. Added KNO2 to reach final conc. of 50 mg N/l, and CH 3COONa to reach final conc. of 300 mg COD/l. Added KNO 3 to reach final conc. of 50 mg N/l. Added KNO3 to reach final conc. of 50 mg N/l, and CH 3COONa to reach final conc. of 300 mg COD/l. Added KNO 3 to final conc. of 50 mg N/l, and CH 3COONa to final conc. of 300 mg COD/l.

End of 2 nd aerobic stage

P NO2

End of 2 nd aerobic stage

P NO3

End of 2 nd aerobic stage

Added NH 4Cl to reach final conc. of 50 mg N/l. Added NH4Cl to reach final conc. of 50 mg N/l, and CH3COONa to reach final conc. of 50 mg COD/l. Added NH 4Cl to reach final conc. of 50 mg N/l. Added NH 4Cl to reach final conc. of 50 mg N/l.

Added CH 3COONa to reach final conc. of 600 mg COD/l, then aeration after 3 hours of phosphate release. Added CH 3COONa to reach final conc. of 600 mg COD/l. After 3 hours of phosphate release, added KNO 2 to reach final conc. of 50 mg N/l. Added CH 3COONa to reach final conc. of 600 mg COD/l. After 3 hours for phosphate release, added KNO 3 to reach final conc. of 50 mg N/l.

ic

ob (3

Effluent

er

Peristaltic pump

na

Pressure gauge

A

Stirrer

SB 2 nd MBR Ae wi rob thd ic ( raw 4h / rs)

Sludge wasted/Influent 11:00 Backwash 23:00 (5 min.)

s)

hr

The aeration time plot of the SBMBR process

s)

0 7:0 0 0 19:

Membrane

2:00 14:00

Aerator

An

ic

(2

st

1

hrs

)

r Ae

hr

i

ob

ox

3 c(

5:00 17:00 (b)

(a)

Fig. 1 (a) Schematic diagram and (b) operating conditions of the SBMBR process

experiments, 2.9 liters of sludge were taken after the anaerobic and anoxic stages of the SBMBR and put into a 3-liter cylinder. A predetermined amount of stock NH 4 Cl solution was added to give an initial concentration of 50 mg NH4-N/l. For the heterotrophic nitrification test (HN-1), extra acetate was added to

give an initial concentration of 50 mg COD/l. In addition, in order to confirm that the nitrite accumulation was due to microbial diversity rather than oxygen limitation, the activated sludge was diluted 4 times to decrease the MLSS concentration (AN-3). For evaluation of the anoxic denitrification performance, 2.9 liters of

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Journal of the Chinese Institute of Engineers, Vol. 33, No. 4 (2010)

Table 2 PCR primer sequences Primer 11f 968fgc 1392r 1512r

Sequence

Reference

GTTTGATCCTGGCTCAG CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGAACGC GAAGAACCTTAC ACGGGCGGTGTGTAC GG(TC)TACCTTGTTACGACTT

sludge were taken after the 1 st and 2 nd aerobic stages of SBMBR and put into a 3-liter circular batch reactor fitted with a screw-down lid. Depending on the different experimental objectives, a predetermined amount of acetate, KNO2, or KNO3 stock solution was added to reach 300 mg COD/l, 50 mg NO2-N/l, or 50 mg NO 3-N/l, respectively, as shown in Table 1. In preparation for the experiments, the samples were taken, filtered, and analyzed rapidly at 0, 1, 10, 20, 30, 45, 60, 90, 120, 180, 240, and 300 minutes.

Amann et al., 1995 Heuer et al., 1997 Ferris et al., 1996 Amann et al., 1995

batch experiments, were rapidly filtered using Whatman GF/A filter paper, after which the NO 2 , NO 3, PO 4 and NH4 contents were analyzed by an ion chromatograph (Dionex ICS-1000, IONPAC column AS12A for anion and CS12A for cation). The suspended solids (SS), chemical oxygen demand (COD), organic nitrogen and total phosphorus (TP) were analyzed according to the Standard Methods: 2540D, 5520, 4500-NorgB, and 4500-PE (APHA, 1989). 4. Analysis of Microbial Diversity

(ii) Batch Experiments for Anaerobic Phosphate Release and Aerobic/Anoxic Phosphate Uptake For the anaerobic phosphate release and aerobic/ anoxic phosphate uptake batch experiments, 2.9 liters of sludge were taken at the end of the 2nd aerobic stage of the SBMBR process and put into a 3-liter circular batch reactor fitted with a screw-down lid. After adding enough predetermined acetate stock solution to reach a COD of 600 mg COD/l, the sludge was homogenized. It remained in the anaerobic phase for phosphate release and COD uptake for 3 hours. Samples were taken at the following times: 0, 1, 5, 15, 30, 45, 60, 90, 120, and 180 minutes. The mixture was then centrifuged at 3000 rpm for 5 minutes. After centrifuging, the supernatant along with un-reacted acetate and released phosphate was decanted, and the concentrated sludge, with intracellular polymer/polyphosphate but without soluble COD or phosphate was resuspended with ddH2O to produce a final volume of 3 liters. The sludge was further divided into 3 sets of 1-liter cylinder batch reactors to perform the aerobic/anoxic phosphate uptake experiments. As shown in Table 1, a certain amount of stock KH 2PO 4 solution was then added to a prereleased PO4-P concentration. For the anoxic condition, enough KNO3 and KNO2 stock solution was added to produce final concentrations of 50 mg NO 3-N/l and 50 mg NO 3 -N/l, respectively. To produce aerobic conditions, enough air was injected into the mixture so that the dissolved oxygen would be more than 2 mg/l. The sampling times were the same as for the anaerobic phosphate-release experiments. 3. Analytical Methods The SBMBR process was monitored every day for 215 days. All samples, including those taken from

In this study, the traditional culturing method was used to identify the microbial diversity in the SBMBR activated sludge. The sludge taken from the SBMBR process was cultured and isolated with TSA (Tryptic soy agar medium: 15.0 g trypton, 5.0 g soytone, 5.0 g NaCl, and 15.0 g agar in 1-liter water) at 35°C for 48 hours in an oven. The DNA of the colonies was further extracted after three cycles of freezing (30 min at -20°C) and thawing (10 min at 95°C) and then amplified with 968fgc and 1392r primer for further denaturing gradient gel electrophoresis (DGGE) examination. Table 2 shows the PCR primer sequences used in this research. The screening was performed with a Biorad Dcode system at 200V for 3 hr. After a silver staining procedure the DGGE patterns were examined. Based on the electrophoresis in the DGGE gel, clones with a unique DGGE electrophoresis position were obtained. These were sequenced with primer 11f using a DNA auto-sequencer (ABI model 377A). The sequences were then compared to the known 16S rRNA sequences in the Genebank using the NCBI BLAST program. III. RESULTS AND DISCUSSION 1. Pilot Plant Performance Table 3 shows the average results from SBMBR daily monitoring for the 215 day operating period. The removal efficiencies of SS, COD, TN and TP of the SBMBR process were found to be 96.7, 95.5, 73.6 and 98.1%, respectively. The removal performance was similar to other MBR processes, but better than traditional biological nutrient removal processes (Stephenson, 2000). It was also found that

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W. S. Chou et al.: Performance of a Sequential Batch Membrane Bio-Reactor for Nutrient Removal

Table 3 Average removal performance of the SBMBR process during the 215 day operating period

(mgSS/l) (mgCOD/l) (mg N/l) (mg N/l) (mg N/l) (mg N/l) (mg N/l) (mg P/l) (mg P/l) (mg P/l)

Average

Range

Effluent Average

Avg. Removal Efficiency (%)

28.5~72.0 508~684 75.0~87.2 33.2~67.4 20.4~37.4 ND* ND 9.0~11.2 1.5~3.4 6.3~9.0

49.0 625 80.2 48.6 31.8 ND ND 10.5 2.1 8.4

ND~3.5 16.0~40.8 13.3~29.9 ND ND 13.3~29.9 ND ND~0.9 ND ND~0.9

1.6 28.0 21.2 ND ND 21.2 ND 0.2 ND 0.2

96.7 95.5 73.6 100 100 — — 98.1 100 97.6

all organic- or ammonia-nitrogen was nitrified into oxidized nitrogen. In addition, it was observed that the nitrite, rather than the nitrate, was accumulated during the operation period. On average, 21.2 mg NO 2 -N/l was detected as the predominant nitrogen species during the SBMBR process. This reveals that partial nitrification had certainly occurred during the SBMBR. Thus, this process proved its ability to lower oxygen consumption for nitrification, for organic compounds that need denitrification and also sludge production, as described earlier (Metcalf and Eddy, 2003). Figure 2 shows the phosphate uptake during 1 st aerobic stage. At the end of the anaerobic stage, 136 mg PO 4-P/l had been released. This had completely been taken-up within 2.5 hours in the following 1 st aerobic stage, corresponding to the phosphate uptake rate of 54.4 mg PO 4-P/l . hr or a specific phosphate uptake rate of 6.55 mg PO 4 -P/gMLSS . hr. It was also observed that no nitrite or nitrate was accumulated in the first 2.5 hours of the 1 st aerobic stage. After phosphate was completely taken-up after the first 2.5 hours of the 1 st aerobic stage, nitrite started to accumulate. This implies that during the first 2.5 hours of the 1 st aerobic stage, when the ammonia was nitrified into oxidized nitrogen, oxidized nitrogen as well as oxygen was consumed simultaneously during phosphate uptake (Carvalho et al., 2007). Table 3 shows that the organic- and ammonianitrogen in the influent were, on average, 48.6 and 31.8 mg N/l respectively. The ammonia-nitrogen decreased from 46.32 to 36.85 mg N/l in the 1 st aerobic stage. In addition, the influent organic-nitrogen underwent further hydrolysis into ammonia-nitrogen in the anaerobic and 1 st aerobic stages. Although the exact organic-nitrogen hydrolysis data can not be measured due to the interference of biomass in the reactor, it is believed that a certain part of the organic-nitrogen was hydrolyzed during the first 6 hours of reaction time in the anaerobic and 1 st aerobic

NH4-N

50

NO2-N

PO4-P

150

40

120

30

90

20

60

10

30

0

0

3

6 Time (hour)

8

12

Phosphate (mgP/l)

SS COD TN Org-N NH 4-N NO 2-N NO 3-N TP Org-P PO 4-P

Range

Ammonia or nitrite (mgN/l)

Influent

Item

0

Fig. 2 Nitrite accumulation in the SBMBR process

stages. Thus, more than 9.47 mg N/l of reduced nitrogen was consumed during the 1 st aerobic stages, probably due to cell growth, heterotrophic nitrification or aerobic denitrification. 2. Nitrification Batch Experiments In order to confirm heterotrophic nitrification, several aerobic nitrification batch experiments were performed in this study as described in Table 1 and the results are shown in Fig. 3. The NH4-N decreased from 55.5 to 49.5 for AN-1 and 56.9 to 47.9 mg N/l for AN-2 within 6 hours of the start of nitrification. The activated sludge was taken from the end of the anaerobic and anoxic stages respectively. Fig. 3(b) shows the specific ammonia oxidizing rates for AN1 and AN-2 were 0.37 and 0.85 mg N/g MLVSS . hr after the first hour, and 0.21 and 0.14 mg N/g MLVSS . hr for the overall 6 hours. This indicates that the nitrification rate was not high, as the nitrite accumulation in the SBMBR process was 5.8 and 7.5 mg N/ l of nitrite in AN-1 and AN-2 batch experiments, respectively. In this study, the average concentration of MLSS for both AN-1 and AN-2 batch experiments was about 8,000 mg/l and the DO was only 1.5 mg/l. There were two probable reasons for the low nitrification rate and

Fig. 3

60.0

AN-1

HN-1

AN-2

AN-3

55.0 50.0 45.0 40.0 35.0 30.0

0

50

100

150 200 250 Time (min)

300

350

400

Nitrite or nitrate concentrations (mgN/l)

Journal of the Chinese Institute of Engineers, Vol. 33, No. 4 (2010)

60.0 ADN1 (Anaerobic, NO2) HDN1 (Anaerobic, NO2) HDN2 (Anoxic, NO2) ADN2 (Anaerobic, NO3) HDN3 (Anaerobic, NO3) HDN4 (Anoxic, NO3)

50.0 40.0 30.0 20.0 10.0 0.0

0

50

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(a) 2.0 AN-1

1.5

HN-1

AN-2

1.0 0.5 0.0

0

1

2

3 Time (hr) (b)

4

5

6

Autotrophic/heterotrophic nitrification batch experiments: (a) ammonia decrease and (b) specific ammonia oxidizing rate

nitrite accumulation, i.e., DO limitation and microbial diversity shift. To identify the influence of DO, another nitrification batch test AN-3 was performed with diluted activated sludge. For the AN-3 test, the DO was kept higher than 4.0 mg/l throughout the experimental batch period. Fig. 3(a) shows the NH 4-N decreased from 56.7 to 50.4 mg N/l within 6 hours, a lower nitrification performance than that of AN-1. In addition, 5.8 mg N/l of nitrite still accumulated as the predominant oxidized nitrogen in the AN-3 test. This indicates that the nitrite accumulation might be due to a shift in microbial diversity after long-term operation under low DO conditions. In addition, it was observed from the heterotrophic nitrification test HN-1 that there was higher heterotrophic nitrification than autotrophic nitrification; see Fig. 3(a). The NH 4 -N decreased from 55.7 to 33.3 mg N/l within 6 hours, corresponding to rates of 1.84 for the first 3 hours, and 0.49 mg N/g MLVSS . hr within 6 hours. It should also be noted that the COD also decreased from 53.8 mg/l to 12.4 mg/l in the first 3 hours, and further to 8.4 mg/l after 6 hours. In addition, no phosphate was present during the experimental period, implying that most of the ammonia was consumed by ammonia oxidization rather than cell growth. On the other hand, 16.4 mg N/l of nitrite was accumulated at the end of the HN-1 experiment, while 22.4 mg N/l of ammonia was consumed. This indicates that 6.0 mg N/l of ammonia might be

Specific denitrifiaction rate (mgN/g MLVSS . hr)

Specific ammonia oxidizing rate (mgN/g MLVSS . hr)

Ammonia concentration (mgN/l)

586

Fig. 4

150 200 250 Time (min) (a)

7.0

300

350

400

ADN1 (Anaerobic, NO2) HDN1 (Anaerobic, NO2) HDN2 (Anoxic, NO2) ADN2 (Anaerobic, NO3) HDN3 (Anaerobic, NO3) HDN4 (Anoxic, NO3)

6.0 5.0 4.0 3.0 2.0 1.0 0.0

0

1

2

3 Time (hr) (b)

4

5

Autotrophic/heterotrophic denitrification batch experiments: (a) nitrite or nitrate decrease and (b) specific denitrification rate

simultaneously oxidizing into nitrite and further denitrifying into nitrogen gas. In our previous study (You and Chen, 2008), we obtained a Bacillus krulwichiaelike species, which contained both ammonia monooxygenase and nitrite reductase genes from the SBMBR which performed ammonia oxidization and nitrite reduction reactions simultaneously. Thus, the SBMBR showed a simultaneous nitrification/denitrification at the 1 st aerobic stage which was proved by the operation of the pilot plant, batch experiments and microbial diversity analysis. 3. Denitrification Batch Experiments Figure 4 shows the results of the autotrophic/ heterotrophic denitrification batch experiments as described in Table 1. As expected, the heterotrophic denitrification rate was obviously higher than the autotrophic denitrification rate, as shown in Fig. 4(a). After heterotrophic denitrification, the initial nitrite or nitrate concentrations for HDN1, HDN2, HDN3 and HDN4 were 50.65, 51.59, 51.36 and 51.69 mg N/l, respectively, while the concentrations after 1 hour of operation were 0.12, 0.22, 0.30, and 0.23 mg N/l respectively, corresponding to specific denitrification rates of 5.81, 6.02, 6.05, and 6.14 mg N/g MLVSS . hr as shown in Fig. 4(b). This revealed that the denitrification rates were almost the same, regardless of whether nitrite or nitrate was used as

6

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the electron acceptor. For autotrophic denitrification, on the other hand, the initial nitrite and nitrate concentrations of ADN1 and ADN2 were 51.82 and 49.62 mg N/l, respectively, while the concentrations after 1 hour were 32.51 and 34.95 mg N/l, and the concentrations after 5 hours were 0.70 and 0.73 mg N/l, respectively, corresponding to specific denitrification rates of 2.14 and 1.93 mg N/g MLVSS . hr for the first hour, and 0.95 and 1.08 mg N/g MLVSS . hr after 5 hours, as shown in Fig. 4(b). The specific autotrophic denitrification rate was only 33.9% that of heterotrophic denitrification. However autotrophic denitrification did occur in both nitrite and nitrate electron acceptor batch experiments. A similar phenomenon was also observed in other studies (Shin et al., 2005; Fabbricino and Petta, 2007). However, no nitrite variation was observed in the last 2 hours of the SBMBR process; as shown in Fig. 2. This reveals that the “autotrophic denitrification” phenomenon mentioned above was likely caused by bacteria which could use intracellular carbon as the source for denitrification. This was not caused by a real autotrophic denitrifier which could use inorganic carbon for denitrification (Bernat and Wojnowska-Baryla, 2007). Many kinds of bacteria, including denitrifying phosphate accumulating organisms (Carvalho et al., 2007; Parco et al., 2007), can consume intracellular polymers for denitrification. Such bacteria can store intracellular carbon sources, even under anaerobic conditions, further oxidizing during denitrification for certain metabolic purposes. 4. Phosphate Release/Uptake Batch Experiments In this study we further tested the phosphate release/uptake potential of the SBMBR activated sludge described in Table 1. As shown in Fig. 5, 211.1 mg/ l of phosphate was released within 3 anaerobic reactions, corresponding to a specific phosphate release rate of 8.80 mg PO 4-P/g MLVSS . hr. The activated sludge samples were centrifuged, the supernatant was decanted, the samples were resuspended in ddH 2O to 3 liters, and finally divided into 3 1-liter cylinder batch sets with oxygen, nitrite and nitrate electron acceptors. It was observed that 68.6, 49.5, and 49.9 mg PO 4-P/l were taken up after the first hour, corresponding to specific phosphate uptake rates of 8.58, 6.19, and 6.24 mg PO4-P/g MLVSS . hr. The phosphate uptake rate was high when oxygen was used as the electron acceptor rather than NOx (Saito et al., 2004). There was no significant difference in the phosphate uptake rate between the nitrite and nitrate electron acceptors. It can also be observed in Fig. 5 that when the nitrite was exhausted after 240 minutes, phosphate release rather than phosphate uptake occurred. Fig. 5 shows that 29.5 mg PO 4 -P/l was released in the

Concentraction (mg N/l or mg P/l)

W. S. Chou et al.: Performance of a Sequential Batch Membrane Bio-Reactor for Nutrient Removal

Nitrite exhausted

250 200

Nitrate exhausted

150 PO4-P (using O2) PO4-P (using NO2) PO4-P (using NO3) NO2-N NO3-N

100 50 0 0

Fig. 5

50

100

150 200 250 Time (min)

300

350

400

Phosphate release/uptake batch experiments using nitrite, nitrate and oxygen as electron acceptors

following two hours. In addition, the nitrate was then exhausted after 300 minutes. However, no phosphate release was observed. Both nitrite and nitrate were considered to be electron acceptors for anoxic phosphate uptake, and the phosphate uptake rate by using nitrate as electron acceptor was faster than using nitrite (Ahn et al., 2001). However, in our experiments, the phosphate uptake rates using nitrite and nitrate as electron acceptors were almost the same. This might be due to the nitrite being the predominant type of oxidized nitrogen in this SBMBR process. The microbial community, including denitrifying phosphate accumulating organisms, was thus accustomed to nitrite as the electron acceptor. 5. Process Optimization According to the above results, the following process optimization was made. (i) For phosphorus removal batch experiment, it was observed that 91.9, 95.7 and 100% of phosphate were release during 90, 120 and 180 minutes based on the total phosphate release at 180 minutes. This showed that it takes 30 and 60 minutes for only 3.1 and 4.3% additional phosphate release over the 1 hour release level. On the other hand, the phosphate removal performance of SBMBR showed that the maximum phosphate release occurred at 150 minutes. The process needs to be optimized to 2.5 hrs of anaerobic RT which will be enough for efficient phosphate release. (ii) In addition, no further phosphate was taken up by the SBMBR process during 150 to 180 minutes, which reveals that 2.5 hrs of aerobic retention time was also enough for phosphate uptake in the 1 st aerobic stage. (iii) On the contrary, it was observed that lots of NH4N was removed without oxidized nitrogen formation during 0 to 120 minutes of the 1st aerobic stage. The consumption of ammonia should be due to the growth of biomass but not nitrification.

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Journal of the Chinese Institute of Engineers, Vol. 33, No. 4 (2010)

Table 4 16S rDNA analysis of nutrient removal bacteria using both pure cultured and cloning methods Clone no. CYCU0255 CYCU0259 CYCU0261 CYCU0265 CYCU0267

Phylogenetic relationship Species

Length (bp)

% of similarity

Bacillus krulwichiae Pedobacter sp. Thauera sp. Acidovorax sp. Aquaspirillum metamorphum

1,275 1,294 1,271 1.248 1,204

96.0 91.5 99.3 96.3 96.0

However, at least 10 mg/l of nitrite was observed from 120 to 180 minutes of the 1st aerobic stage. If aerobic time was reduced to 30 minutes, as measured by phosphate uptake, the nitrogen removal performance might be worse. Thus, 3 hrs of 1 st aerobic retention time is still needed. (iv) For the anoxic stage, only nitrite reduction was observed during the first 75 minutes, this revealed that 45 minutes can be saved in the anoxic stage. (v) For the 2 nd aerobic stage, it was observed that less than 4 mg/l ammonia was further nitrified into nitrite during 120 to 240 minutes. This seems to show that the 2nd aerobic retention time can be shorted to 2 hrs. However, it should be noticed that the MBR was drained out during the 2nd aerobic stage. If the 2nd aerobic retention time was shortened, the volume of effluent was decreased, resulting in a higher HRT. (vi) According to the above discussion, 30 to 45 minutes can be taken from the anaerobic and anoxic stages, respectively, and 75 minutes added to the 2 nd aerobic stage. This also results in increased effluent volume (31.25%) under similar HRT operating conditions. 6. Nutrient-Removal Related Bacterial Analysis We also analyzed the microbial diversity of the SBMBR sludge using both traditional culturing and gene cloning methods. For the traditional culturing method, 150 colonies were picked and then screened by DGGE. 12 different OTUs were obtained and their 16S rRNA genes were further sequenced. After comparision with the Genebank of NCBI, 4 of these 12 sequences were identified as nutrient removal related bacteria, as shown in Table 4. The Bacillus krulwichiae detected contained both ammonia monooxygenase and nitrite reductase genes, found in our previous study to perform ammonia oxidization and nitrite reduction reactions simultaneously (You and Chen, 2008). Pedobacter sp. is a kind of Gram negative rod bacteria. It has already been isolated from a nitrifying inoculum (Vanparys et al., 2001). Shinoda and his-coworkers (2004) have examined Thauera sp..

They found that it can degrade toluene by using nitrate as an electron acceptor. They further considered Thauera sp. to be a kind of denitrifier which was also confirmed by Thomsen et al. (2007). We also isolated the Acidovorax sp. which can accumulate granular PHB and reduce nitrate. It is considered as denitrifying phosphate accumulating bacteria (Schulze et al., 1999). The genera of Aquaspirillum, as well as Azoarcus, Thauera, and Rhodocyclus are considered to be the predominant denitrifiers. They made up 2049% of all bacteria from 17 nitrogen removal wastewater treatment plants (Thomsen et al., 2007). IV. CONCLUSIONS In this study, we performed a nitrite accumulating SBMBR process and identified the microbial diversity of the activated sludge. The SBMBR showed excellent nutrient removal efficiency. The nitrite accumulating phenomenon might have occurred due to a shift in the microbial diversity after long-term operation under low DO conditions. The results of the batch experiments also showed autotrophic denitrification and heterotrophic nitrification. The phosphate uptake rate was significantly better when oxygen was used as an electron acceptor rather than NOx; there was no significant difference in phosphate uptake rate observed between nitrite and nitrate as the electron acceptors. The microbial diversity of the SBMBR sludge was analysed using traditional culturing methods. Several candidate nutrient removal bacteria were isolated, such as Pedobacter sp., Bacillus krulwichiae, Thauera sp., Acidovorax sp., and Aquaspirillum metamorphum. The results reveal high microbial diversity in this reactor. ACKNOWLEDGMENTS This study was supported by a grant received from the National Science Council of the Republic of China (No. NSC 94-2211-E-033-003). REFERENCES Ahn, J., Daidou, T., Tsuneda, S., and Hirata, A., 2001,

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