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Behavior of total phosphorus removal in an intelligent controlled sequencing batch biofilm reactor for municipal wastewater treatment
Bioresource Technology 132 (2013) 190–196
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Behavior of total phosphorus removal in an intelligent controlled sequencing batch biofilm reactor for municipal wastewater treatment Wei Cai a,b, Baogang Zhang a,b,⇑, Yunxiao Jin c, Zhongfang Lei d, Chuanping Feng a,b,⇑, Dahu Ding d, Weiwu Hu a,b, Nan Chen a,b, Takashi Suemura e a
Key Laboratory of Groundwater Circulation and Evolution (China University of Geosciences Beijing), Ministry of Education, Beijing 100083, China School of Water Resources and Environment, China University of Geosciences Beijing, Beijing 100083, China Department of Civil Engineering, Luoyang Institute of Science and Technology, No. 90 Wangcheng Road, Luoyang 471023, China d Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan e Advanced Logic Systems Company, Tsukuba, Ibaraki 305-8572, Japan b c
h i g h l i g h t s " Phosphorus is successfully removed in sequencing batch biofilm reactor (SBBR). " SBBR controlled by an intelligent control system (ICS) performs better. " No significant anaerobic phosphorus release is observed in ICS-SBBR. " Polyhydroxyalkanoates (PHAs) is the main energy source. " Biochemical metabolism of phosphorus removal in ICS-SBBR is elucidated.
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Article history: Received 23 October 2012 Received in revised form 26 December 2012 Accepted 27 December 2012 Available online 7 January 2013 Keywords: Sequencing batch biofilm reactor Phosphorus removal Polyhydroxyalkanoates Glycogen Energy metabolism
a b s t r a c t The behavior of total phosphorus removal was investigated in present study in sequencing batch biofilm reactor (SBBR) controlled by an intelligent control system (ICS) with less energy consumption for municipal wastewater treatment. Stable total phosphorus (TP) removal efficiency of 93.9 ± 2.2% was achieved in comparison to that of 93.3 ± 2.5% in a conventional timer control system (TCS-SBBR). Significant anaerobic phosphorus release was not observed in ICS-SBBR, which was unlike the conventional TCS-SBBR. Moreover, lower accumulations/transformations of polyhydroxyalkanoates (PHAs) and higher transformation of glycogen occurred in the ICS-SBBR, indicating that PHAs was the main energy source while glycogen played a supporting role when PHAs were inadequate, which was different from the traditional mechanism of biological phosphorus removal in TCS-SBBR. The possible biochemical metabolism of phosphorus removal in ICS-SBBR was also elucidated. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Phosphorus is a well known nutrient element necessary for biological growth (Morse et al., 1998), while excessive discharge of phosphorus may cause eutrophication in surface water bodies like lakes and reservoirs (Ugurlu and Salman, 1998). This can lead to reduction in biological diversity and recreational value of natural water bodies, loss of livestock and human health issues (Mullan et al., 2006). Thus it is necessary to remove phosphate from wastewater before discharge. ⇑ Corresponding authors. Address: School of Water Resources and Environment, China University of Geosciences Beijing, Beijing 100083, China Tel.: +86 10 8232 2281; fax: +86 10 8232 1081. E-mail addresses: [email protected] (B. Zhang), [email protected] (C. Feng). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.181
Various techniques have been developed for phosphorus removal, such as adsorption, chemical precipitation, crystallization and biological phosphorus removal (Morse et al., 1998; Ding et al., 2012; Chen et al., 2012; Liu et al., 2012; Xu et al., 2012; Coats et al., 2011). Among these methods, biological phosphorus removal is considered as the most economical and sustainable process and has been widely adapted in real operations for phosphorus removal plants (Jeon and Park, 2000). Up to now activated sludge processes like anoxic/oxic (A/O), anaerobic/anoxic/oxic (A/A/O) and sequencing batch reactor (SBR) are playing major roles in the aspect of biological phosphorus removal (Oehmen et al., 2007). However, these processes are prone to unpredictable failures due to biomass loss (Oehmen et al., 2007; Blackall et al., 2002). Biofilm processes possess many advantages, including land and energy savings, greater biomass concentration, flexible operation, lower sensitivity to toxicity and greater volumetric loads (Rodgers and
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Zhan, 2003), which are very attractive to many researchers. For example, the capabilities of biological aerated filter (BAF) (Chen et al., 2011) and sequencing batch biofilm reactor (SBBR) (Fu et al., 2010) for phosphorus removal have been investigated. As the operation mode plays an important role in SBBR applications, our research group has developed an intelligent control systemSBBR (ICS-SBBR) to remove phosphorus with further energy saving and simplified operation (Ding et al., 2011; Jin et al., 2012). Theories for biological phosphorus removal have been established and developed since the phenomenon first being reported that activated sludge could take up excessive phosphorus required for normal biomass growth (Shrinath et al., 1959). The mechanism of enhanced biological phosphorus removal (EBPR) process has been mostly studied. The EBPR process mainly bases on activated sludge which is enriched with polyphosphate accumulating organisms (PAOs). PAOs can release phosphorus during anaerobic phase and then take up and transform phosphorus as polyphosphate during aerobic phase (Oehmen et al., 2007) and net removal of phosphorus can be achieved via discharging activated sludge rich in poly-P. Specifically, the EBPR process links the cycling of two kinds of intracellular organic carbon storage polymers (PHAs and glycogen) to phosphorus removal (Coats et al., 2011). However, the mechanism of phosphorus removal by PAOs is not fully established under conditions that differ from the classical anaerobic/aerobic conditions (Pijuan et al., 2005) and some phenomena cannot be explained by the existing mechanism. For example, Wang et al. (2009) demonstrated that the efficiency of phosphorus removal could be increased using glucose as the sole carbon source under single-stage oxic conditions. Kapagiannidis et al. (2012) reported that phosphorus removal also occurred in a continuous-flow anaerobic–anoxic (A2) activated sludge system. In our previous research (Jin et al., 2012), phosphorus removal could be realized without significant anaerobic phosphorus release, indicating that there might be a special pattern for phosphorus removal. In this study, an intelligent control system (ICS), based on both DO concentration and temperature in the wastewater, was adopted to control an SBBR to treat raw wastewater. An SBBR controlled by time control system (TCS) was also introduced as the control sets. The performances of phosphorus removal in the two SBBRs were investigated. Carbon and energy sources such as PHAs and glycogen in both two SBBRs were also monitored in order to study the mechanism of phosphorus removal in ICS-SBBR. 2. Methods 2.1. Inoculation and the raw wastewater The sludge seed used in this study were obtained from the reclaimed water station of Tsinghua University, Beijing, China. The mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) of the activated sludge seed were 9870 and 6570 mg/l, respectively. The value of MLVSS/MLSS was 0.67. The raw wastewater was collected from the students’ dormitory and cafeteria and was characterized in Table 1. 2.2. Reactor configurations Experiments were carried out in two SBBRs controlled by an intelligent control system (ICS) and a timer control system (TCS),
respectively. As shown in Fig. 1, both reactors were made of polymethyl methacrylate, with the dimensions of 400 250 300 mm (L W H) and a working volume of 20 L. Hundred porous polyacrylonitrile balls (38 mm of diameter) were chosen as biofilm carriers for each reactor with filling ratio about 25% because of their higher specific areas (300 m2/m3) and lower costs.
2.3. Operation procedures Each reactor was inoculated with about 10 L activated sludge seed first, and then the SBBRs were operated at the hydraulic retention time of 12 h for biofilms formation and activated sludge domestication. The air flow rate was controlled at 300 l/h. When a layer of biofilm that adhered on the filler was visible to the naked eye, start-up period was assumed to be achieved in the two SBBRs. After the start-up period, the cycles of two SBBRs were set at 8 h (FILL 0.3 h, RUN 7 h, SETTLE 0.5 h, and DRAW 0.2 h). In every DRAW phase, the planktonic microbes above the outlet were discharged, while others remained in reactors. The total operation duration lasted 60 days after the start-up. Operation in present study was divided into two phases based on temperature, the first phase started (0–50 d, 22–25 °C) and the second phase (51–60 d, 16–18 °C), where the temperature fluctuation was caused by seasonal change. The operating procedure of the ICS-SBBR ran as follow: the RUN phase contained one calculation period (1 h) and three reaction periods (6 h). First, the calculation period contained an aeration phase (0.5 h) and a non-aeration phase (0.5 h). Next, the ICS produced a schedule of aeration and non-aeration time based on the temperature and the respiration rate of microorganisms, which were measured every 2 h (Ding et al., 2011). While the TCS-SBBR was operated with 3 h anaerobic phase followed by 4 h aeration phase controlled by a timer. Fig. 2 showed a typical cycle of the ICS-SBBR. The typical principle of the three reaction periods in the ICS-SBBR was as follows, [aeration (85 min) + no aeration (anaerobic, 45 min)] ? [aeration (60 min) + no aeration (anaerobic, 60 min)] ? [aeration (25 min) + no aeration (anaerobic, 95 min)].
2.4. Analytical methods Water samples were collected once a day at the end of one cycle and all water samples were filtered with 0.45 lm membrane. During the total operation cycle, samples were obtained at the time point when the aerobic phase and anaerobic phase alternated in ICS-SBBR; and in the TCS-SBBR samples were taken every hour. Samples collected from the reactors were immediately centrifuged and the supernatant was analyzed for chemical oxygen demand (COD) and total phosphorus (TP), and the filtered matter was assayed for MLSS, MLVSS, PHAs and glycogen. The temperature and DO were measured continuously online using a DO detector (HACH sc100, USA). The pH was measured with a portable pH meter (Mettler Toledo S02, USA). Turbidity was measured by a portable turbidimeter (HACH 2100P, USA). COD was analyzed by a HACH DR/890 colorimeter. TP was determined by ultraviolet–visible spectrophotometer (Shimadzu UV2450, Japan) according to Standard Methods (American Public Health Association, 2005). MLSS and MLVSS were also determined
Table 1 Characteristics of the raw wastewater during the stable operation of 60 days. Parameter
COD (mg/l)
NH3-N (mg/l)
TN (mg/l)
TP (mg/l)
pH
Turbidity (NTU)
BOD5/COD
Conductivity (mS/m)
Values
294.1 ± 87.0
47.9 ± 9.1
63.2 ± 10.7
5.53 ± 0.88
7.04 ± 0.08
133.9 ± 32.6
0.4–0.6
165.3 ± 96.7
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Fig. 1. Schematic diagrams of ICS-SBBR (a) and TCS-SBBR (b). (1. ICS; 2. DO controller; 3. DO probe; 4. circulating pump; 5. emptying pipe; 6. aeration panel; 7. porous polyacrylonitrile balls; 8. flow meter; 9. air compressor circulating pump; 10. sampling ports; 11.TCS.)
Fig. 2. A typical operation cycle of the ICS-SBBR (the abscissa represents reaction time; the vertical axis represents the air flow of aeration).
according to Standard Methods (American Public Health Association, 2005). Glycogen in the sludge sample was measured using a phenol method (Randall and Liu, 2002). The PHAs (poly-3-hydroxybutyrate (PHB) and poly-3-hydroxyvalerate (PHV)) were analyzed using a gas chromatography (GC) (Shimadzu GC-2014, Japan) (Oehmen et al., 2005). All samples were lyophilized first, and then acidified methanol solution and chloroform were added to digest at 100 °C, followed by cooling and mixing with water to achieve phase separation and the chloroform phase was quantified using GC, operated with a DB-5 column (30 m length 0.25 mm I.D. 1.40 lm film), a split injection ratio of 1:10 and helium as the carrier gas (1.5 ml/min). A flame ionization detection (FID) unit was operated at 250 °C with an injection port temperature of 230 °C. The oven temperature was set to 80 °C for 1 min and increased at 8 °C/min to 120 °C, then by 30 °C/min to 220 °C and held for 2 min.
3. Results and discussion 3.1. TP removal during continuous operation Fig. 3 showed that TP could be removed effectively during the stable operation period after the domestication in the two SBBRs. During the first phase, the TP concentration in the influent was 5.53 ± 0.88 mg/l. While stable TP removals were achieved in the first phase for the two SBBRs, with effluent TP concentrations being 0.33 ± 0.11 mg/l and 0.36 ± 0.12 mg/l in the ICS-SBBR and TCSSBBR, respectively. The corresponding removal efficiencies were 93.9 ± 2.2% and 93.3 ± 2.5%, respectively. Both of them showed high efficiency with respect to TP removal, which was similar to the previous research (Fu et al., 2010) while slightly lower than
the result (>94.1% for synthetic wastewater treatment) obtained by Jin et al. (2012), possibly caused by the fluctuated loading of phosphorus (Lu et al., 2007). This observation indicated that the two SBBRs were effective for phosphorus pollution control. In addition, the performance of the ICS-SBBR was slightly better than the TCS-SBBR, showing its superior adaptability to loading fluctuation. From Fig. 3 the TP removal performance for the two SBBRs were observed to become worse at the beginning of the second phase (from 50 to 55 d) mainly due to the sudden drop of temperature (from 25 °C to 16 °C), and then re-stabilized (from 56 to 60 d). During the period of 50–55 d, the effluent TP increased to 0.74 ± 0.18 and 1.67 ± 0.48 mg/l with the corresponding removal efficiencies decreased to 90.3 ± 2.7% and 86.0 ± 8.1% for ICS-SBBR and TCSSBBR, respectively, indicating that temperature had significant influence on biological phosphorus removal by affecting the activities of PAOs (Mulkerrins et al., 2004). Ren et al. (2011) also found the phosphate removal efficiency decreased from 59.3% to 52.1% in an EBPR system when the temperature changed from 29 to 14 °C. However, the TP removal efficiencies in ICS-SBBR and TCS-SBBR recovered to 94.0 ± 0.9% and 91.9 ± 2.5% during the 56–60 d, respectively, indicating that the PAOs could restore their strong activities after a short adaptation period even under low temperature conditions (16–18 °C). The result was in agreement with the observation of Li et al. (2010) who obtained the highest TP removal efficiency of 95% at 15 °C after adaption. Some investigations suggested that lower temperature favored the growth of PAOs over other microorganisms (Oehmen et al., 2007), and the formation of more intracellular polymers PHB which made the PAOs had stronger competitive capability when the added external substrates were exhausted during aerobic phase (Oehmen et al., 2007). Besides, TP concentration in the effluent from ICS-SBBR was more stable compared with that from TCS-SBBR, implying that the adaptability of PAOs to different operation conditions was
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Fig. 3. Phosphorus removals in two SBBRs during 60 days’ stable operation period. First phase, 0–50 days; second phase, 50–60 days.
enhanced due to the frequent change of aerobic and anaerobic periods in the ICS-SBBR. 3.2. Characteristics of pollutants removal and energy conversion in total operating cycle The characteristics of ICS-SBBR and TCS-SBBR in a total operating cycle were investigated during steady operation. For the ICSSBBR, the aeration time during reaction period was getting shorter and shorter (from 75 min to 60 min and to 25 min, shown in Fig. 2), completed by the ICS which could automatically adjust the aeration time with intelligence and flexibility according to the oxygen demand and the real-time DO concentration in the wastewater (Ding et al., 2011). While in the TCS-SBBR, the aeration time was fixed at 4 h which was longer than ICS-SBBR, indicating that the ICS-SBBR consumed less energy compared with the TCS-SBBR for raw wastewater treatment (Jin et al., 2012). 3.2.1. Concentration variations of COD and TP The concentration variations of TP and COD during the total operation cycle in the ICS-SBBR and TCS-SBBR were shown in Fig. 4. In ICS-SBBR, COD concentration rapidly decreased from 370 to 51 mg/l during the first aerobic 30 min (Fig. 4a). However, the value re-increased to 190 mg/l after the anaerobic in calculation period. It could be deduced that the COD reduction during the initial 30 min was mainly through initial physical adsorption (Jin et al., 2012). The increase in COD concentration afterward was caused by desorption due to the change of ICS-SBBR from aerobic to anaerobic condition. Thereafter, the COD concentration in
the ICS-SBBR decreased gradually, meeting the Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant in China (GB 18918-2002) (less than 50 mg/l) in 180 min. The result also showed that during the first aerobic 30 min the TP concentration rapidly decreased from 6.03 to 3.52 mg/l in ICSSBBR (Fig. 4a), which was different from the previous findings that the existence of abundant substrates could restrain phosphorus uptake under aerobic environment (Kuba et al., 1994). It was proposed that most of the decreased phosphate was mainly through absorption in this period. Unlike COD behavior, no desorption occurred during the TP removal process. Afterwards, a relatively steady TP decrease was observed during the remaining operation cycle with TP concentration gradually decreased below the Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant in China (GB 18918-2002) (less than 0.5 mg/l) after 120 min. It should be stressed that no obvious TP release was observed in anaerobic phase before TP uptake in aerobic phase during the four times of aerobic/anaerobic alternation, except a slight increase to 1.11 mg/l after the second anaerobic phase, suggesting there might be some specific mechanisms of TP removal in the ICS-SBBR. Fig. 4b showed the COD and TP variations during standard anaerobic/aerobic cycle in the TCS-SBBR. In the first 30 min of anaerobic phase, the COD concentration decreased sharply from 370 to 169 mg/l, mainly contributed by adsorption (Dulekgurgen et al., 2003). Desorption, however, did not occur in the TCS-SBBR possibly owing to the long duration of anaerobic condition (180 min), during which the external carbon had been transformed into intracellular carbon polymers (PHAs and glycogen) subsequently serving as carbon source for phosphorus uptake and
Fig. 4. The variations of TP and COD during a total operation cycle. (a) ICS-SBBR (b) TCS-SBBR.
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denitrification after the anaerobic phase. The degradation of COD required 240 min in the TCS-SBBR. During the anaerobic phase, the TP concentration in the TCSSBBR increased from initial 6.03 to 19.33 mg/l, and then sharply decreased to a low level (0.58 mg/l) during the aerobic phase due to the phosphorus uptake by PAOs. The TP removal behavior in the TCS-SBBR was similar to the conventional EBPR process in which phosphorus was significantly released by PAOs during anaerobic phase while rapidly absorbed for PAOs growth and intracellular poly-P formation in aerobic phase (Oehmen et al., 2007). Stable phosphorus removal performances were both achieved in ICS-SBBR and TCS-SBBR. It is known that C, N and P are required in proportions (e.g. C:N:P = 100:10:1 for wastewater) for maintaining microorganisms growth and reproduction (Yu et al., 2007). In present study, the ratios of Cuptake/Puptake were about 50 and excess phosphorus removal was observed in both ICS-SBBR and TCS-SBBR. However, in contrast to TCS-SBBR, no significant phosphorus release was observed in the ICS-SBBR during the anaerobic phase of the total cycle. It was proposed that the variations of intracellular PHAs and glycogen during the phosphorus removal process in ICS-SBBR were quite different from the conventional TCS-SBBR, which would be discussed in the next part.
3.2.2. Variations of PHAs and glycogen The intracellular storage (PHAs and glycogen) responsible for phosphorus removals during the total operation cycle were also monitored in the two SBBRs, which exhibited different variation principles. In ICS-SBBR (Fig. 4a), PHAs were rapidly accumulated within the first 30 min aeration (about 0.65 mmol-C/g VSS), indicating that PHAs could also be synthesized under aeration phase (Fig. 4a), which was consistent with the results observed by other researchers (Wang et al., 2009; Carta et al., 2001). The result also showed that the storage of PHB mainly contributed to the PHAs accumulation with average ratio of PHBaccumulated/PHAsaccumulated about 88.9%. PAOs are reported to produce PHB chiefly from acetate and PHV mainly from propionate (Oehmen et al., 2005). Therefore, it could be deduced that the acetate were firstly taken up in ICSSBBR, and then the accumulated PHAs were gradually decomposed until the end of the cycle except a slight increase due to the PHV accumulation from 135 to 180 min coupled with the increase of TP, indicating that acetate in the wastewater was exhausted and other volatile fatty acids (VFAs) (main propionate) was consumed during this period. Glycogen concentration almost had no change within the first 30 min in ICS-SBBR (Fig. 4a), indicating that glycolysis did not occur during this period. After that, the glycogen started to be accumulated (about 2.11 mmol-C/g VSS) along with the decomposing of PHAs from 30 min to 180 min. This observation might be re-
sulted from the fact that the energy for glycogen synthesis was provided by PHAs decomposition during this period, in accordance with the finding of Wang et al. (2009). After this period, the glycogen concentration decreased gradually until the end of operating cycle, indicating that glycogen was used to provide energy for cell growth when COD decreased to a very low level (Fig. 4a). In the TCS-SBBR, the initial PHAs concentration was about 1.00 mmol-C/g VSS and increased along with glycogen degradation during the first 60 min, implying that glycogen decomposed to provide energy for the synthesis of PHAs through glycolysis (Fig. 5b). Then small amount of PHAs were consumed and glycogen concentration gradually returned to its initial level at 180 min. In aerobic phase, the PHAs were gradually decomposed while the glycogen content almost had no change, denoting that PHAs were the main energy source for phosphorus removal and cell growth and maintenance.
3.3. Energy metabolism of phosphorus removal The variation of COD and TP as well as PHAs and glycogen indicated two different behaviors in phosphorus removal between ICSSBBR and TCS-SBBR. Fig. 6 showed the proposed metabolisms in ICS-SBBR and TCS-SBBR, with respect to phosphate uptake and energy storages formations. A stable phosphorus removal performance was realized in the total cycle of ICS-SBBR although no significant anaerobic phosphorus release was observed; different from the TCS-SBBR in which most energy substances were accumulated during the process of anaerobic phosphorus release (Oehmen et al., 2007). In the ICSSBBR, PHAs were the main energy while glycogen could play a supporting role when the PHAs content was inadequate. It has been demonstrated that PHAs is a kind of reduced polymer and its synthesis requires a reducing equivalent source, such as reduced nicotinamide adenine dinucleotide (NADH) (Mino et al., 1998). Two possible pathways were proposed to explain the source of the reducing power, i.e. the Comeau-Wentzel model suggested partial oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle generated the reducing equivalents; and in the Mino model reducing power was derived from glycolysis of intracellular stored glycogen (Mino et al., 1998; Louie et al., 2000). In the ICS-SBBR, glycogen concentration remained almost unchanged with the accumulation of PHAs indicating the reducing power of PHAs synthesis did not come from glycogen decomposition but possibly from the oxidation of acetyl-CoA and propionyl-CoA through the TCA cycle corresponded with the Comeau-Wentzel model (Fig. 6a). According to the Comeau-Wentzel model, the PHAs storage comes from acetyl-CoA and propionyl-CoA, and the precursor for PHB formation is 2 acetyl-CoA and PHV formation requires the above two precursors in equal amounts (Wang et al., 2009). From
Fig. 5. The variations of PHAs (PHB and PHV) and glycogen during a total operation cycle. (a) ICS-SBBR (b) TCS-SBBR.
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Fig. 6. The energy metabolism of poly-P organisms in the ICS-SBBR (a) and TCS-SBBR (b).
Fig. 5a, about 0.16 mmol-C/g VSS PHB and 0.03 mmol-C/g VSS PHV were accumulated within 30-min aeration, so the required acetylCoA and propionyl-CoA was 7.55 mg/g VSS and 1.32 mg/g VSS, respectively, which might be converted from the acetate and propionate adsorbed from the wastewater. When it came to anaerobic phase, the PHAs were decomposed to provide energy to remove phosphorus and synthesize glycogen. In the ICS-SBBR, the results of PHAs degradation and the glycogen accumulation indicated that PHAs were the primary energy sources during phosphorus removal process and when the PHAs content decreased to low levels, glycogen would be decomposed as a main energy source for cell growth instead. This phenomenon was similar with Wang et al. who noticed that glycogen was rapidly decomposed to provide maintenance energy when the added external substrates exhausted (Wang et al., 2009). In the TCS-SBBR, the phosphorus removal behavior was similar to conventional EBPR. During the anaerobic phase, PAOs could take up VFAs in wastewater and store them as intracellular carbon polymers PHAs (Fig. 6b). The required reducing equivalents source (NADH) for the synthesis of PHAs under anaerobic conditions was produced largely through the glycolysis of internally stored glycogen which was accord with Mino model (Mino et al., 1998). During the aerobic phase, PAOs mainly utilized the intracellular PHAs as carbon and energy sources to enable normal metabolism and generated energy for phosphorus uptake, synthesis of polyphosphate and cell growth (Mullan et al., 2006; Blackall et al., 2002).
4. Conclusions A stable TP removal performance (93.9 ± 2.2%) with no significant anaerobic phosphorus release was achieved in ICS-SBBR, which had less energy consumption compared with the result (93.3 ± 2.5%) obtained in TCS-SBBR. The ICS-SBBR adapted to the temperature fluctuation more effectively in contrast to TCS-SBBR. Furthermore, PHAs were the main energy and glycogen played a supporting role when the PHAs content was inadequate in ICSSBBR, while the PHAs were the main energy substrate and the glycogen only provides energy or intermediate for the synthesis of PHAs in TCS-SBBR. The possible biochemical metabolism of phosphorus removal in ICS-SBBR was proposed and the distinguishing principle was also elucidated.
Acknowledgements Financial support is gratefully acknowledged from the National Natural Science Foundation (No. 31140082) and the Fundamental Research Funds for the Central Universities (2011YYL109).
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Behavior of total phosphorus removal in an intelligent controlled sequencing batch biofilm reactor for municipal wastewater treatment Wei Cai a,b, Baogang Zhang a,b,⇑, Yunxiao Jin c, Zhongfang Lei d, Chuanping Feng a,b,⇑, Dahu Ding d, Weiwu Hu a,b, Nan Chen a,b, Takashi Suemura e a
Key Laboratory of Groundwater Circulation and Evolution (China University of Geosciences Beijing), Ministry of Education, Beijing 100083, China School of Water Resources and Environment, China University of Geosciences Beijing, Beijing 100083, China Department of Civil Engineering, Luoyang Institute of Science and Technology, No. 90 Wangcheng Road, Luoyang 471023, China d Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan e Advanced Logic Systems Company, Tsukuba, Ibaraki 305-8572, Japan b c
h i g h l i g h t s " Phosphorus is successfully removed in sequencing batch biofilm reactor (SBBR). " SBBR controlled by an intelligent control system (ICS) performs better. " No significant anaerobic phosphorus release is observed in ICS-SBBR. " Polyhydroxyalkanoates (PHAs) is the main energy source. " Biochemical metabolism of phosphorus removal in ICS-SBBR is elucidated.
a r t i c l e
i n f o
Article history: Received 23 October 2012 Received in revised form 26 December 2012 Accepted 27 December 2012 Available online 7 January 2013 Keywords: Sequencing batch biofilm reactor Phosphorus removal Polyhydroxyalkanoates Glycogen Energy metabolism
a b s t r a c t The behavior of total phosphorus removal was investigated in present study in sequencing batch biofilm reactor (SBBR) controlled by an intelligent control system (ICS) with less energy consumption for municipal wastewater treatment. Stable total phosphorus (TP) removal efficiency of 93.9 ± 2.2% was achieved in comparison to that of 93.3 ± 2.5% in a conventional timer control system (TCS-SBBR). Significant anaerobic phosphorus release was not observed in ICS-SBBR, which was unlike the conventional TCS-SBBR. Moreover, lower accumulations/transformations of polyhydroxyalkanoates (PHAs) and higher transformation of glycogen occurred in the ICS-SBBR, indicating that PHAs was the main energy source while glycogen played a supporting role when PHAs were inadequate, which was different from the traditional mechanism of biological phosphorus removal in TCS-SBBR. The possible biochemical metabolism of phosphorus removal in ICS-SBBR was also elucidated. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Phosphorus is a well known nutrient element necessary for biological growth (Morse et al., 1998), while excessive discharge of phosphorus may cause eutrophication in surface water bodies like lakes and reservoirs (Ugurlu and Salman, 1998). This can lead to reduction in biological diversity and recreational value of natural water bodies, loss of livestock and human health issues (Mullan et al., 2006). Thus it is necessary to remove phosphate from wastewater before discharge. ⇑ Corresponding authors. Address: School of Water Resources and Environment, China University of Geosciences Beijing, Beijing 100083, China Tel.: +86 10 8232 2281; fax: +86 10 8232 1081. E-mail addresses: [email protected] (B. Zhang), [email protected] (C. Feng). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.181
Various techniques have been developed for phosphorus removal, such as adsorption, chemical precipitation, crystallization and biological phosphorus removal (Morse et al., 1998; Ding et al., 2012; Chen et al., 2012; Liu et al., 2012; Xu et al., 2012; Coats et al., 2011). Among these methods, biological phosphorus removal is considered as the most economical and sustainable process and has been widely adapted in real operations for phosphorus removal plants (Jeon and Park, 2000). Up to now activated sludge processes like anoxic/oxic (A/O), anaerobic/anoxic/oxic (A/A/O) and sequencing batch reactor (SBR) are playing major roles in the aspect of biological phosphorus removal (Oehmen et al., 2007). However, these processes are prone to unpredictable failures due to biomass loss (Oehmen et al., 2007; Blackall et al., 2002). Biofilm processes possess many advantages, including land and energy savings, greater biomass concentration, flexible operation, lower sensitivity to toxicity and greater volumetric loads (Rodgers and
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Zhan, 2003), which are very attractive to many researchers. For example, the capabilities of biological aerated filter (BAF) (Chen et al., 2011) and sequencing batch biofilm reactor (SBBR) (Fu et al., 2010) for phosphorus removal have been investigated. As the operation mode plays an important role in SBBR applications, our research group has developed an intelligent control systemSBBR (ICS-SBBR) to remove phosphorus with further energy saving and simplified operation (Ding et al., 2011; Jin et al., 2012). Theories for biological phosphorus removal have been established and developed since the phenomenon first being reported that activated sludge could take up excessive phosphorus required for normal biomass growth (Shrinath et al., 1959). The mechanism of enhanced biological phosphorus removal (EBPR) process has been mostly studied. The EBPR process mainly bases on activated sludge which is enriched with polyphosphate accumulating organisms (PAOs). PAOs can release phosphorus during anaerobic phase and then take up and transform phosphorus as polyphosphate during aerobic phase (Oehmen et al., 2007) and net removal of phosphorus can be achieved via discharging activated sludge rich in poly-P. Specifically, the EBPR process links the cycling of two kinds of intracellular organic carbon storage polymers (PHAs and glycogen) to phosphorus removal (Coats et al., 2011). However, the mechanism of phosphorus removal by PAOs is not fully established under conditions that differ from the classical anaerobic/aerobic conditions (Pijuan et al., 2005) and some phenomena cannot be explained by the existing mechanism. For example, Wang et al. (2009) demonstrated that the efficiency of phosphorus removal could be increased using glucose as the sole carbon source under single-stage oxic conditions. Kapagiannidis et al. (2012) reported that phosphorus removal also occurred in a continuous-flow anaerobic–anoxic (A2) activated sludge system. In our previous research (Jin et al., 2012), phosphorus removal could be realized without significant anaerobic phosphorus release, indicating that there might be a special pattern for phosphorus removal. In this study, an intelligent control system (ICS), based on both DO concentration and temperature in the wastewater, was adopted to control an SBBR to treat raw wastewater. An SBBR controlled by time control system (TCS) was also introduced as the control sets. The performances of phosphorus removal in the two SBBRs were investigated. Carbon and energy sources such as PHAs and glycogen in both two SBBRs were also monitored in order to study the mechanism of phosphorus removal in ICS-SBBR. 2. Methods 2.1. Inoculation and the raw wastewater The sludge seed used in this study were obtained from the reclaimed water station of Tsinghua University, Beijing, China. The mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) of the activated sludge seed were 9870 and 6570 mg/l, respectively. The value of MLVSS/MLSS was 0.67. The raw wastewater was collected from the students’ dormitory and cafeteria and was characterized in Table 1. 2.2. Reactor configurations Experiments were carried out in two SBBRs controlled by an intelligent control system (ICS) and a timer control system (TCS),
respectively. As shown in Fig. 1, both reactors were made of polymethyl methacrylate, with the dimensions of 400 250 300 mm (L W H) and a working volume of 20 L. Hundred porous polyacrylonitrile balls (38 mm of diameter) were chosen as biofilm carriers for each reactor with filling ratio about 25% because of their higher specific areas (300 m2/m3) and lower costs.
2.3. Operation procedures Each reactor was inoculated with about 10 L activated sludge seed first, and then the SBBRs were operated at the hydraulic retention time of 12 h for biofilms formation and activated sludge domestication. The air flow rate was controlled at 300 l/h. When a layer of biofilm that adhered on the filler was visible to the naked eye, start-up period was assumed to be achieved in the two SBBRs. After the start-up period, the cycles of two SBBRs were set at 8 h (FILL 0.3 h, RUN 7 h, SETTLE 0.5 h, and DRAW 0.2 h). In every DRAW phase, the planktonic microbes above the outlet were discharged, while others remained in reactors. The total operation duration lasted 60 days after the start-up. Operation in present study was divided into two phases based on temperature, the first phase started (0–50 d, 22–25 °C) and the second phase (51–60 d, 16–18 °C), where the temperature fluctuation was caused by seasonal change. The operating procedure of the ICS-SBBR ran as follow: the RUN phase contained one calculation period (1 h) and three reaction periods (6 h). First, the calculation period contained an aeration phase (0.5 h) and a non-aeration phase (0.5 h). Next, the ICS produced a schedule of aeration and non-aeration time based on the temperature and the respiration rate of microorganisms, which were measured every 2 h (Ding et al., 2011). While the TCS-SBBR was operated with 3 h anaerobic phase followed by 4 h aeration phase controlled by a timer. Fig. 2 showed a typical cycle of the ICS-SBBR. The typical principle of the three reaction periods in the ICS-SBBR was as follows, [aeration (85 min) + no aeration (anaerobic, 45 min)] ? [aeration (60 min) + no aeration (anaerobic, 60 min)] ? [aeration (25 min) + no aeration (anaerobic, 95 min)].
2.4. Analytical methods Water samples were collected once a day at the end of one cycle and all water samples were filtered with 0.45 lm membrane. During the total operation cycle, samples were obtained at the time point when the aerobic phase and anaerobic phase alternated in ICS-SBBR; and in the TCS-SBBR samples were taken every hour. Samples collected from the reactors were immediately centrifuged and the supernatant was analyzed for chemical oxygen demand (COD) and total phosphorus (TP), and the filtered matter was assayed for MLSS, MLVSS, PHAs and glycogen. The temperature and DO were measured continuously online using a DO detector (HACH sc100, USA). The pH was measured with a portable pH meter (Mettler Toledo S02, USA). Turbidity was measured by a portable turbidimeter (HACH 2100P, USA). COD was analyzed by a HACH DR/890 colorimeter. TP was determined by ultraviolet–visible spectrophotometer (Shimadzu UV2450, Japan) according to Standard Methods (American Public Health Association, 2005). MLSS and MLVSS were also determined
Table 1 Characteristics of the raw wastewater during the stable operation of 60 days. Parameter
COD (mg/l)
NH3-N (mg/l)
TN (mg/l)
TP (mg/l)
pH
Turbidity (NTU)
BOD5/COD
Conductivity (mS/m)
Values
294.1 ± 87.0
47.9 ± 9.1
63.2 ± 10.7
5.53 ± 0.88
7.04 ± 0.08
133.9 ± 32.6
0.4–0.6
165.3 ± 96.7
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Fig. 1. Schematic diagrams of ICS-SBBR (a) and TCS-SBBR (b). (1. ICS; 2. DO controller; 3. DO probe; 4. circulating pump; 5. emptying pipe; 6. aeration panel; 7. porous polyacrylonitrile balls; 8. flow meter; 9. air compressor circulating pump; 10. sampling ports; 11.TCS.)
Fig. 2. A typical operation cycle of the ICS-SBBR (the abscissa represents reaction time; the vertical axis represents the air flow of aeration).
according to Standard Methods (American Public Health Association, 2005). Glycogen in the sludge sample was measured using a phenol method (Randall and Liu, 2002). The PHAs (poly-3-hydroxybutyrate (PHB) and poly-3-hydroxyvalerate (PHV)) were analyzed using a gas chromatography (GC) (Shimadzu GC-2014, Japan) (Oehmen et al., 2005). All samples were lyophilized first, and then acidified methanol solution and chloroform were added to digest at 100 °C, followed by cooling and mixing with water to achieve phase separation and the chloroform phase was quantified using GC, operated with a DB-5 column (30 m length 0.25 mm I.D. 1.40 lm film), a split injection ratio of 1:10 and helium as the carrier gas (1.5 ml/min). A flame ionization detection (FID) unit was operated at 250 °C with an injection port temperature of 230 °C. The oven temperature was set to 80 °C for 1 min and increased at 8 °C/min to 120 °C, then by 30 °C/min to 220 °C and held for 2 min.
3. Results and discussion 3.1. TP removal during continuous operation Fig. 3 showed that TP could be removed effectively during the stable operation period after the domestication in the two SBBRs. During the first phase, the TP concentration in the influent was 5.53 ± 0.88 mg/l. While stable TP removals were achieved in the first phase for the two SBBRs, with effluent TP concentrations being 0.33 ± 0.11 mg/l and 0.36 ± 0.12 mg/l in the ICS-SBBR and TCSSBBR, respectively. The corresponding removal efficiencies were 93.9 ± 2.2% and 93.3 ± 2.5%, respectively. Both of them showed high efficiency with respect to TP removal, which was similar to the previous research (Fu et al., 2010) while slightly lower than
the result (>94.1% for synthetic wastewater treatment) obtained by Jin et al. (2012), possibly caused by the fluctuated loading of phosphorus (Lu et al., 2007). This observation indicated that the two SBBRs were effective for phosphorus pollution control. In addition, the performance of the ICS-SBBR was slightly better than the TCS-SBBR, showing its superior adaptability to loading fluctuation. From Fig. 3 the TP removal performance for the two SBBRs were observed to become worse at the beginning of the second phase (from 50 to 55 d) mainly due to the sudden drop of temperature (from 25 °C to 16 °C), and then re-stabilized (from 56 to 60 d). During the period of 50–55 d, the effluent TP increased to 0.74 ± 0.18 and 1.67 ± 0.48 mg/l with the corresponding removal efficiencies decreased to 90.3 ± 2.7% and 86.0 ± 8.1% for ICS-SBBR and TCSSBBR, respectively, indicating that temperature had significant influence on biological phosphorus removal by affecting the activities of PAOs (Mulkerrins et al., 2004). Ren et al. (2011) also found the phosphate removal efficiency decreased from 59.3% to 52.1% in an EBPR system when the temperature changed from 29 to 14 °C. However, the TP removal efficiencies in ICS-SBBR and TCS-SBBR recovered to 94.0 ± 0.9% and 91.9 ± 2.5% during the 56–60 d, respectively, indicating that the PAOs could restore their strong activities after a short adaptation period even under low temperature conditions (16–18 °C). The result was in agreement with the observation of Li et al. (2010) who obtained the highest TP removal efficiency of 95% at 15 °C after adaption. Some investigations suggested that lower temperature favored the growth of PAOs over other microorganisms (Oehmen et al., 2007), and the formation of more intracellular polymers PHB which made the PAOs had stronger competitive capability when the added external substrates were exhausted during aerobic phase (Oehmen et al., 2007). Besides, TP concentration in the effluent from ICS-SBBR was more stable compared with that from TCS-SBBR, implying that the adaptability of PAOs to different operation conditions was
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Fig. 3. Phosphorus removals in two SBBRs during 60 days’ stable operation period. First phase, 0–50 days; second phase, 50–60 days.
enhanced due to the frequent change of aerobic and anaerobic periods in the ICS-SBBR. 3.2. Characteristics of pollutants removal and energy conversion in total operating cycle The characteristics of ICS-SBBR and TCS-SBBR in a total operating cycle were investigated during steady operation. For the ICSSBBR, the aeration time during reaction period was getting shorter and shorter (from 75 min to 60 min and to 25 min, shown in Fig. 2), completed by the ICS which could automatically adjust the aeration time with intelligence and flexibility according to the oxygen demand and the real-time DO concentration in the wastewater (Ding et al., 2011). While in the TCS-SBBR, the aeration time was fixed at 4 h which was longer than ICS-SBBR, indicating that the ICS-SBBR consumed less energy compared with the TCS-SBBR for raw wastewater treatment (Jin et al., 2012). 3.2.1. Concentration variations of COD and TP The concentration variations of TP and COD during the total operation cycle in the ICS-SBBR and TCS-SBBR were shown in Fig. 4. In ICS-SBBR, COD concentration rapidly decreased from 370 to 51 mg/l during the first aerobic 30 min (Fig. 4a). However, the value re-increased to 190 mg/l after the anaerobic in calculation period. It could be deduced that the COD reduction during the initial 30 min was mainly through initial physical adsorption (Jin et al., 2012). The increase in COD concentration afterward was caused by desorption due to the change of ICS-SBBR from aerobic to anaerobic condition. Thereafter, the COD concentration in
the ICS-SBBR decreased gradually, meeting the Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant in China (GB 18918-2002) (less than 50 mg/l) in 180 min. The result also showed that during the first aerobic 30 min the TP concentration rapidly decreased from 6.03 to 3.52 mg/l in ICSSBBR (Fig. 4a), which was different from the previous findings that the existence of abundant substrates could restrain phosphorus uptake under aerobic environment (Kuba et al., 1994). It was proposed that most of the decreased phosphate was mainly through absorption in this period. Unlike COD behavior, no desorption occurred during the TP removal process. Afterwards, a relatively steady TP decrease was observed during the remaining operation cycle with TP concentration gradually decreased below the Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant in China (GB 18918-2002) (less than 0.5 mg/l) after 120 min. It should be stressed that no obvious TP release was observed in anaerobic phase before TP uptake in aerobic phase during the four times of aerobic/anaerobic alternation, except a slight increase to 1.11 mg/l after the second anaerobic phase, suggesting there might be some specific mechanisms of TP removal in the ICS-SBBR. Fig. 4b showed the COD and TP variations during standard anaerobic/aerobic cycle in the TCS-SBBR. In the first 30 min of anaerobic phase, the COD concentration decreased sharply from 370 to 169 mg/l, mainly contributed by adsorption (Dulekgurgen et al., 2003). Desorption, however, did not occur in the TCS-SBBR possibly owing to the long duration of anaerobic condition (180 min), during which the external carbon had been transformed into intracellular carbon polymers (PHAs and glycogen) subsequently serving as carbon source for phosphorus uptake and
Fig. 4. The variations of TP and COD during a total operation cycle. (a) ICS-SBBR (b) TCS-SBBR.
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denitrification after the anaerobic phase. The degradation of COD required 240 min in the TCS-SBBR. During the anaerobic phase, the TP concentration in the TCSSBBR increased from initial 6.03 to 19.33 mg/l, and then sharply decreased to a low level (0.58 mg/l) during the aerobic phase due to the phosphorus uptake by PAOs. The TP removal behavior in the TCS-SBBR was similar to the conventional EBPR process in which phosphorus was significantly released by PAOs during anaerobic phase while rapidly absorbed for PAOs growth and intracellular poly-P formation in aerobic phase (Oehmen et al., 2007). Stable phosphorus removal performances were both achieved in ICS-SBBR and TCS-SBBR. It is known that C, N and P are required in proportions (e.g. C:N:P = 100:10:1 for wastewater) for maintaining microorganisms growth and reproduction (Yu et al., 2007). In present study, the ratios of Cuptake/Puptake were about 50 and excess phosphorus removal was observed in both ICS-SBBR and TCS-SBBR. However, in contrast to TCS-SBBR, no significant phosphorus release was observed in the ICS-SBBR during the anaerobic phase of the total cycle. It was proposed that the variations of intracellular PHAs and glycogen during the phosphorus removal process in ICS-SBBR were quite different from the conventional TCS-SBBR, which would be discussed in the next part.
3.2.2. Variations of PHAs and glycogen The intracellular storage (PHAs and glycogen) responsible for phosphorus removals during the total operation cycle were also monitored in the two SBBRs, which exhibited different variation principles. In ICS-SBBR (Fig. 4a), PHAs were rapidly accumulated within the first 30 min aeration (about 0.65 mmol-C/g VSS), indicating that PHAs could also be synthesized under aeration phase (Fig. 4a), which was consistent with the results observed by other researchers (Wang et al., 2009; Carta et al., 2001). The result also showed that the storage of PHB mainly contributed to the PHAs accumulation with average ratio of PHBaccumulated/PHAsaccumulated about 88.9%. PAOs are reported to produce PHB chiefly from acetate and PHV mainly from propionate (Oehmen et al., 2005). Therefore, it could be deduced that the acetate were firstly taken up in ICSSBBR, and then the accumulated PHAs were gradually decomposed until the end of the cycle except a slight increase due to the PHV accumulation from 135 to 180 min coupled with the increase of TP, indicating that acetate in the wastewater was exhausted and other volatile fatty acids (VFAs) (main propionate) was consumed during this period. Glycogen concentration almost had no change within the first 30 min in ICS-SBBR (Fig. 4a), indicating that glycolysis did not occur during this period. After that, the glycogen started to be accumulated (about 2.11 mmol-C/g VSS) along with the decomposing of PHAs from 30 min to 180 min. This observation might be re-
sulted from the fact that the energy for glycogen synthesis was provided by PHAs decomposition during this period, in accordance with the finding of Wang et al. (2009). After this period, the glycogen concentration decreased gradually until the end of operating cycle, indicating that glycogen was used to provide energy for cell growth when COD decreased to a very low level (Fig. 4a). In the TCS-SBBR, the initial PHAs concentration was about 1.00 mmol-C/g VSS and increased along with glycogen degradation during the first 60 min, implying that glycogen decomposed to provide energy for the synthesis of PHAs through glycolysis (Fig. 5b). Then small amount of PHAs were consumed and glycogen concentration gradually returned to its initial level at 180 min. In aerobic phase, the PHAs were gradually decomposed while the glycogen content almost had no change, denoting that PHAs were the main energy source for phosphorus removal and cell growth and maintenance.
3.3. Energy metabolism of phosphorus removal The variation of COD and TP as well as PHAs and glycogen indicated two different behaviors in phosphorus removal between ICSSBBR and TCS-SBBR. Fig. 6 showed the proposed metabolisms in ICS-SBBR and TCS-SBBR, with respect to phosphate uptake and energy storages formations. A stable phosphorus removal performance was realized in the total cycle of ICS-SBBR although no significant anaerobic phosphorus release was observed; different from the TCS-SBBR in which most energy substances were accumulated during the process of anaerobic phosphorus release (Oehmen et al., 2007). In the ICSSBBR, PHAs were the main energy while glycogen could play a supporting role when the PHAs content was inadequate. It has been demonstrated that PHAs is a kind of reduced polymer and its synthesis requires a reducing equivalent source, such as reduced nicotinamide adenine dinucleotide (NADH) (Mino et al., 1998). Two possible pathways were proposed to explain the source of the reducing power, i.e. the Comeau-Wentzel model suggested partial oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle generated the reducing equivalents; and in the Mino model reducing power was derived from glycolysis of intracellular stored glycogen (Mino et al., 1998; Louie et al., 2000). In the ICS-SBBR, glycogen concentration remained almost unchanged with the accumulation of PHAs indicating the reducing power of PHAs synthesis did not come from glycogen decomposition but possibly from the oxidation of acetyl-CoA and propionyl-CoA through the TCA cycle corresponded with the Comeau-Wentzel model (Fig. 6a). According to the Comeau-Wentzel model, the PHAs storage comes from acetyl-CoA and propionyl-CoA, and the precursor for PHB formation is 2 acetyl-CoA and PHV formation requires the above two precursors in equal amounts (Wang et al., 2009). From
Fig. 5. The variations of PHAs (PHB and PHV) and glycogen during a total operation cycle. (a) ICS-SBBR (b) TCS-SBBR.
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Fig. 6. The energy metabolism of poly-P organisms in the ICS-SBBR (a) and TCS-SBBR (b).
Fig. 5a, about 0.16 mmol-C/g VSS PHB and 0.03 mmol-C/g VSS PHV were accumulated within 30-min aeration, so the required acetylCoA and propionyl-CoA was 7.55 mg/g VSS and 1.32 mg/g VSS, respectively, which might be converted from the acetate and propionate adsorbed from the wastewater. When it came to anaerobic phase, the PHAs were decomposed to provide energy to remove phosphorus and synthesize glycogen. In the ICS-SBBR, the results of PHAs degradation and the glycogen accumulation indicated that PHAs were the primary energy sources during phosphorus removal process and when the PHAs content decreased to low levels, glycogen would be decomposed as a main energy source for cell growth instead. This phenomenon was similar with Wang et al. who noticed that glycogen was rapidly decomposed to provide maintenance energy when the added external substrates exhausted (Wang et al., 2009). In the TCS-SBBR, the phosphorus removal behavior was similar to conventional EBPR. During the anaerobic phase, PAOs could take up VFAs in wastewater and store them as intracellular carbon polymers PHAs (Fig. 6b). The required reducing equivalents source (NADH) for the synthesis of PHAs under anaerobic conditions was produced largely through the glycolysis of internally stored glycogen which was accord with Mino model (Mino et al., 1998). During the aerobic phase, PAOs mainly utilized the intracellular PHAs as carbon and energy sources to enable normal metabolism and generated energy for phosphorus uptake, synthesis of polyphosphate and cell growth (Mullan et al., 2006; Blackall et al., 2002).
4. Conclusions A stable TP removal performance (93.9 ± 2.2%) with no significant anaerobic phosphorus release was achieved in ICS-SBBR, which had less energy consumption compared with the result (93.3 ± 2.5%) obtained in TCS-SBBR. The ICS-SBBR adapted to the temperature fluctuation more effectively in contrast to TCS-SBBR. Furthermore, PHAs were the main energy and glycogen played a supporting role when the PHAs content was inadequate in ICSSBBR, while the PHAs were the main energy substrate and the glycogen only provides energy or intermediate for the synthesis of PHAs in TCS-SBBR. The possible biochemical metabolism of phosphorus removal in ICS-SBBR was proposed and the distinguishing principle was also elucidated.
Acknowledgements Financial support is gratefully acknowledged from the National Natural Science Foundation (No. 31140082) and the Fundamental Research Funds for the Central Universities (2011YYL109).
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