Biological phosphorus removal in sequencing batch reactor with single-stage oxic process

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Bioresource Technology 99 (2008) 5466–5473

Biological phosphorus removal in sequencing batch reactor with single-stage oxic process Dong-bo Wang, Xiao-ming Li *, Qi Yang, Guang-ming Zeng, De-xiang Liao, Jie Zhang College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR China Received 2 August 2007; received in revised form 5 November 2007; accepted 5 November 2007 Available online 21 December 2007

Abstract The performance of biological phosphorus removal (BPR) in a sequencing batch reactor (SBR) with single-stage oxic process was investigated using simulated municipal wastewater. The experimental results showed that BPR could be achieved in a SBR without anaerobic phase, which was conventionally considered as a key phase for BPR. Phosphorus (P) concentration 0.22–1.79 mg L 1 in effluent can be obtained after 4 h aeration when P concentration in influent was about 15–20 mg L 1, the dissolved oxygen (DO) was controlled at 3 ± 0.2 mg L 1 during aerobic phase and pH was maintained 7 ± 0.1, which indicated the efficiencies of P removal were achieved 90% above. Experimental results also showed that P was mainly stored in the form of intracellular storage of polyphosphate (poly-P), and about 207.235 mg phosphates have been removed by the discharge of rich-phosphorus sludge for each SBR cycle. However, the energy storage poly-b-hydroxyalkanoates (PHA) was almost kept constant at a low level (5–6 mg L 1) during the process. Those results showed that phosphate could be transformed to poly-P with single-stage oxic process without PHA accumulation, and BPR could be realized in net phosphate removal. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Biological phosphorus removal; Poly-b-hydroxyalkanoates; Poly-phosphate; Sequencing batch reactor

1. Introduction Levin and Shapiro (1965) studied absorption and release of phosphate in the 1960s and advanced the theory of BPR, which considering the opinion that BPR was achieved through Emden–Meyerhof and tricarboxylic acid (TCA) cycle. Researches of Harold (1966), Nicholls and Osborn (1979) developed the theory of BPR. It took the point that poly-P was a response of anaerobic microorganism; furthermore, PHA was a key material for the subsistence of microorganism in anaerobic condition. Wentzel et al. (1989) proposed Comeau/Wentzel model and figured that deoxidization energy was produced through partly oxidization of acetate in TCA circulation. The present traditional theory of BPR was formed gradually based on a large number of research experiments, which considered that BPR is *

Corresponding author. Tel.: +86 731 8823967; fax: +86 731 8822829. E-mail address: [email protected] (X.-m. Li).

0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.11.007

an activated sludge process operated with sequential anaerobic and aerobic phases. A group of bacteria such as Acinetobacter, Pseudomonas (Kim and Pagilla, 2000; Lin et al., 2003) and Candidatus accumulibacter phosphates bacteria (Ahn et al., 2007; Cai et al., 2007) are regarded as representative phosphate accumulating organisms (PAOs) and predominant species to the whole of bacterial population in activated sludge for P removal. PAOs take up volatile fatty acids (VFAs) and convert them to intracellular PHA anaerobically, and gain energy coupled with reducing power required for anaerobic VFAs uptake as well as conversion to PHA through the hydrolysis of their intracellularly stored poly-P and glycogen (van Loosdrecht et al., 1997). Aerobically, PAOs oxidize PHA to gain energy for growth, glycogen replenishment and P uptake. Because the phosphate uptake in aerobic condition is far greater than the released in anaerobic phase, a high performance of P removal can be achieved by the discharge of rich-phosphorus sludge (Pijuan et al., 2005; Harper et al.,

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2006; Mullan et al., 2006). Based on the conventional theory of BPR, many researches mainly about efficiencies improvements and effects of environmental conditions on P removal as well as PAOs’ activities evaluation have reported recently (Kuba et al., 1996; Hu et al., 2002; Merzouki et al., 2005). The main discoveries in past one year are summarized below: complete P removal (100%) was achieved using an influent chemical oxygen demand (COD): P ratio of 15:1 with over 50 mg orthophosphate (PO4-P)/l being consistently removed by Broughton et al. (2008). Li et al. (2008) obtained a high P removal efficiency of 97% with the 2/1 propionic/acetic acid as organic substrate. Chiou and Yang (2007) showed that the anaerobic phase was more significant than the aerobic phase on P storage capability of the PAOs. A model-based evaluation of competition between PAOs and GAOs under different operational conditions was presented by Whang et al. (2007). Carvalho et al. (2007) investigated the link between the process performance of two denitrifying P removal systems and their microbial community structure. Although PAOs such as Acinetobacter are widely accepted as effective bacteria for BPR, there are some other organisms also show the possibility (Kornberg et al., 1999). Lotter and Murphy (1985) have found that Pseudomonas spp., Moraxella spp., Aeromonas spp., etc. could also accumulate poly-P aerobically in activated sludge. Microthrix parvicella (Blackall et al., 1995; Erhart et al., 1997) and Nostocoida limicola (Blackall et al., 2000) have been still found to store poly-P in aerobic phase. Since M. parvicella is able to take up long chain fatty acids under anaerobic conditions and possess a similar P uptake mechanism (Andreasen and Nielsen, 1997, 1998), many researchers suggested that it might belong to the traditional PAOs with a P storage/release mechanism comparable to the one present in microorganisms responsible for BPR (Wanner, 1994; Tandoi et al., 1998). However, M. parvicella can not be a typical PAO as it does not consume acetate under in situ conditions. To date, the microorganisms responsible for BPR are not well identified yet, nor characterized in activated sludge (Mino et al., 1998; Filipe et al., 2001). In this research, it was found that P removal could be achieved without specific anaerobic phase in an inner-loop SBR by our research group at the beginning in October 2005, and then further experiments were conducted in a traditional SBR with single-stage oxic phase from October 2005 to May 2007 to verify this phenomenon. The aim of this paper was to introduce the peculiar P removal phenomenon and to assess P removal efficiencies in SBR with single-stage oxic phase with the use of chemical analytical techniques. 2. Methods 2.1. Experimental device and operational methods Experiments were mainly carried out in SBRs with a working volume of 12 L, which were made of Lucite (the

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barrel diameter 15 cm, working height 70 cm). The 8 h cycles consisted of approximately 4 h aerobic period, 4 h settle/decant/idle period. Synthetic wastewater (composition detailed below) was fed to reactor during the first 5 min of the aerobic period. For each SBR cycle, about 7.8 L aqueous was draw out, which implied the hydraulic retention time (HRT) was maintained approximately 12 h. In this process, anaerobic phase does not exist but long-term settle/decant/idle zone (4 h) is operated during two cycles. In aerobic phase, DO concentration was controlled at 3 ± 0.2 mg L 1 using an on/off control valve that was connected to a compressed air supply. The pH was controlled at 7 ± 0.1 during aerobic period through addition of 0.5 M HCl and 0.5 M NaOH. Activated sludge, taken from the first municipal wastewater treatment plant of Changsha, PR China, was seeded and acclimated according to the way described above, and the initial concentration of sludge in reactor was set around 4000 mg L 1. 2.2. Synthetic media Synthetic wastewater was used in this research. Glucose is used as the carbon source, and phosphate in the wastewater is simulated with potassium dihydrogen phosphate (KDP). The COD concentration in the feed for each SBR was about 400 mg COD/L, while the P concentration was about 20 mg PO4-P/L, which yielded a COD/P ratio of 20 mg COD/mg PO4-P. The concentration of the other nutrients in the synthetic feed are indicated below (per liter): 0.02 g NH4Cl, 0.02 g peptone, 0.01 g MgSO4
7H2O, 0.005 g CaCl2, and 0.5 mL of a trace metals solution. The trace metals solution has also been described in Smolders et al. (1994) and consisted of (per liter): 1.5 g FeCl3
6H2O, 0.15 g H3BO3, 0.03 g CuSO4
5H2O, 0.18 g KI, 0.12 g MnCl2
4H2O, 0.06 g Na2MoO4
2H2O, 0.12 g ZnSO4
7H2O, 0.15 g CoCl2
6H2O and 10 g EDTA. 2.3. Analytical methods Mixed liquor suspended solid (MLSS), mixed liquor volatile suspended solid (MLVSS), COD and PO4-P were measured according to Standard Methods (APHA, 1995). PHA was measured with Gas Chromatography (GC). About 50 mL sludge samples were mixed with formaldehyde at a ratio of approximately 1% formaldehyde per sample volume in order to inhibit biomass activity in the sludge. The samples were centrifuged and the supernatant was removed, and then washed with a phosphate buffer solution, re-centrifuged, and the supernatant decanted once more. All samples were then lyophilized through a freeze drying unit operated at 54 °C and 0.1 mbar for at least 20 h. Approximately 20 mg of lyophilized sludge was added to 2 mL of chloroform and 2 mL of an acidified methanol solution, and then the samples were digested in tightly at 100 °C in an oven for 7 h and cooled to room temperature. Distilled water (2 mL) was then added and

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mixed vigorously with each sample to remove particulate debris from the chloroform phase and prevent degradation of the GC column. After mixing, 1 h of setting time was allowed to achieve phase separation. The chloroform (bottom) phase was then injected into the GC column. The chromatography was operate with a DB-5 column (30 m length  0.25 mm LD  0.25 lm film), a spilt injection ratio of 1:15 and helium as the carrier gas (1.5 mL/min). A flame ionization detection (FID) unit was operated at 300 °C with an injection port temperature of 250 °C. The oven temperature was set to 80 °C for 1 min, increased at 10 °C/min to 120 °C, and then to 270 °C at 45 °C/min and held for 3 min (Randall and Liu, 2002; Oehmen et al., 2005a). Poly-P: about 50 mL sludge samples were filtrated and cleaned with distilled water 3 times, and then dried at 120 °C in an oven. Approximately 30 mg of dried sludge was cleaned and centrifuged with perchloric acid (0.5 mol L 1), 75%, 95% and anhydrous ethanol, respectively. Poly-P were sequentially extracted from biomass by acidic (0.5 M HClO4 at 0 °C), salt (5 mg NaClO4 and 0.005 mg of 1 M HClO4 for every 10 mg of initial sample), and alkaline (cold 0.05 M NaOH) solutions and the following fractions were thus obtained: acid_soluble, salt soluble and alkali_soluble poly-P. Amount of labile P in all poly-P fractions was determined by the orthophosphate content formed as a result of acidic hydrolysis in the presence of 1 M HCl for 10 min at 100 °C. (Buzoleva et al., 2006). 3. Results 3.1. P removal performances during long-term operation The domestication of activated sludge in SBR system for P removal finished after about 14 days, and then experiments for P removal were conducted and lasted for about 180 days. The efficiencies of P removal for each cycle during this period are partly shown in Fig. 1.

As shown in Fig. 1, it can be clearly observed that a poor level of P removal was obtained during the first 7 days, and then P concentrations in effluent decreased gradually. After 27 days, the efficiencies of P removal were steady in SBR. P concentrations in effluent were kept among 0.22–1.79 mg L 1 mostly and the removal efficiencies were above 90%. Since this peculiar phenomenon could not be explained by the conventional BPR theories, 5 times experiments were repeated on P removal in SBR with single-stage oxic process from March 2006 to May 2007, and similar results were obtained. The experiments proved that P removal could be achieved without anaerobic phase, which was conventionally considered as an absolutely necessary phase for BPR. 3.2. P removal and PHA accumulation in a typical cycle As well known that, the anaerobic phase is very important for traditional P removal, intracellular poly-P is hydrolyzed to phosphate and released to wastewater by PAOs. Meanwhile, external carbon sources is converted to PHA as energy storage for the subsequent aerobic phase after influent for BPR (Chen et al., 2005), thus BPR could not be realized without PHA accumulation in an anaerobic zone during the process. Question might be raised would be if the long idle phase served as the anaerobic phase? Fig. 2 showed some datum about P and PHA in a typical cycle after about 90-day steady state operation. In this study, aeration was carried out without anaerobic phase mix after influent wastewater, and PHA monitoring results showed that the energy storage PHA was almost kept constant at a low level (5–6 mg L 1) during the whole process. But P concentration was decreased obviously along with aeration. As can be seen from Fig. 2, P concentration was 19.84 mg L 1 in influent coupled with 0.28 mg L 1 in effluent and the efficiency of P removal reached 98.59%. In the idle period between two cycles, although the system was in anaerobic, carbon substrates

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in wastewater were depleted mostly after 4 h aeration. As a result, little PHA change was observed in this phase. The experimental results indicated that P removal could be achieved with single-stage oxic phase in SBR without energy storage PHA accumulation, which was considered as a key matter for traditional BPR. 3.3. The approach of P removal in this study One possibility for P removal in this study could be that P might be absorbed by activated sludge. Since the SBR systems were long-term running, we argued that if P was removed by sludge adsorption, the saturation would happen soon after the system running, and P, then, would be no longer to be removed in high efficiency. In fact, P were removed in high efficiencies all along after steady state operation, therefore, the possibility could be excluded. There are three forms of P in activated sludge: biological P (bio-P), metal P though physical chemistry process and poly-P (Janssen et al., 2002). Bio-P is necessary for microorganism growth and metabolism, thus another possibility for P removal in this study was that P removal was the result of normal growth and metabolism by microorganism. Janssen et al. (2002) showed that P/TSS was 1–2% in activated sludge which indicated that P incepted by microorganism growth and metabolism was finite. Furthermore, bio-P in activated sludge was associated with COD concentration, and previous investigations have shown that the COD/P rate by normal microorganism growth and metabolism was 200 mg COD/1 mg PO4-P. Since that the COD/P rate in this study was 20 mg COD/ 1 mg PO4-P, which was much lower than the normal COD/P rate (200:1). As a result, we excluded the possibility. PO4-P can be formed metal sediments like MgHPO4, MgNH4PO4, Ca5(PO4)3OH, FePO4 etc. if there are some metal ions like Mg2+, Ca2+, Fe3+ etc. in wastewater. Could P removal in this study be through this approach? Since the Mg2+, Ca2+, Fe2+ concentrations in this study were very low (concentrations detailed above) and the sediments above must be formed under the condition of pH > 7.5

(Janssen et al., 2002), and pH values in this study was controlled at 7 ± 0.1. Therefore, the hypothesis was also excluded. To investigate the approach of P removal in this study, the varieties of poly-P, PO4-P, COD and MLSS during a typical process were analyzed after about 135-day steady state operation, which are shown in Fig. 3. As shown in Fig. 3, a rapid COD concentration decrease was associated with a fast MLSS increase at the first 30 min (COD concentration was from 396 mg L 1 to 66 mg L 1, MLSS concentration was from 4056 mg L 1 to 4220 mg L 1). However, a low amount of P uptake as well as obvious poly-P content decrease was observed (P uptake was only 1.72 mg L 1, poly-P content decrease was 3.347 mg g SS 1), which may be the results of abundant carbon sources existing during this period, previous researches have shown that abundant substrates existing in aerobic could restrain P uptake (Kuba et al., 1994). A high amount of P uptake along with a quick poly-P content increase was obtained from 30 to 90 min, which implied that PO4-P was transformed to intracellular poly-P in this study. After 90 min, P concentration was decreased while intracellular poly-P was increased gradually during aeration. COD and MLSS concentrations have no obvious varieties after 30 min in aeration, and then at the end of aerobic period, COD, MLSS, TP concentrations and poly-P content were 26 mg L 1, 4250 mg L 1, 0.28 mg L 1 and 86.338 mg g SS 1, respectively. For each SBR cycle, about 200 mg L 1 of MLSS (2.4 g MLSS) was drawn out which indicated the sludge retention time (SRT) was maintained approximately 7 days in order to stabilize the MLSS concentration, that means about 207.235 mg phosphates removed by the discharge of rich-phosphorus sludge while about 228 mg phosphates were fed to each cycle in influent, which suggested that P was mainly removed in the form of poly-P. In the settle/decant/idle zone (240–480 min in Fig. 3) between two cycles, since the MLSS was settled at the bottom of the reactor, DO concentrations of the system consisted of two parts (DO in aqueous phase and in settled

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sludge). A low DO decrease in aqueous phase as well as a high DO decrease in settled sludge was observed during the settle/decant/idle zone (DO concentrations in aqueous phase were 3.06 mg L 1 at 240 min, 2.57 mg L 1 at 360 min and 2.53 mg L 1 at 480 min, and DO concentrations in settled sludge were 1.87 mg L 1 at 270 min, 0.86 mg L 1 at 360 min and 0.55 mg L 1 at 480 min). Although the internal system of settled sludge was in anoxic, the settle/decant/idle zone was not the anaerobic phase which we conducted in the conventional anaerobic/ aerobic process. As well known that, in the very low DO condition (generally, DO < 0.5 mg L 1), high COD decrease along with much PHA accumulation and high poly-P hydrolysis as well as much P release was obtained in the anaerobic phase of conventional anaerobic/aerobic process (Akin and Ugurlu, 2004). However, poly-P in the settle/decant/idle zone of this study was not hydrolyzed largely to phosphate as we considered in the past studies (poly-P content decrease was only 0.2 mg g SS 1, and the P release was just 0.83 mg L 1) and a little MLSS decrease was associated with a little COD increase. This phenomenon is also different from traditional studies. 4. Discussion Excess phosphate uptake by bacteria in the activated sludge process was observed for the first time by Vaker et al. (1967). Identification of the dominant bacteria responsible for phosphate removal has been elucidated (Buchan, 1983; Lotter and Murphy, 1985). Some previous workers (Buchan, 1981; Lotter, 1985) have shown that Acinetobacter played an important role in phosphate removal by activated sludge systems. Moreover, many researchers have shown that not only one species but also several of

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bacteria, which existed in activated sludge, could remove P (Gersberg and Alien, 1985; Suresh et al., 1985), and Lin et al. (2003) suggested that P removal capability of activated sludge seemed to be due to the increase in the populations of bacteria with activity of P removal. In traditional anaerobic/aerobic (A/O) process, PAOs can obtain energy from poly-P hydrolyzation and rapidly take up coupled with store substrate under anaerobic conditions. As a result, this mechanism of PAOs gain a strong competitive advantage compared to most other microorganisms including other poly-P organisms in such systems (Andreasen and Nielsen, 2000; Janssen et al., 2002). However, in this study, glucose which was thought to be not good enough for PAOs (Cech and Hartman, 1993) was only used as organic substrate. Additionally, conventional anaerobic period was not conducted and little PHA accumulation as well as little phosphate release was observed during idle phase, P removal in this research was not mainly performed by traditional PAOs but likely by other poly-P organisms such as M. parvicella which conducted P uptake for poly-P in aerobic phase without anaerobic P release and substrates uptake (Janssen et al., 2002). Casey et al. (1992, 1994) and Xue et al. (2007) considered that poly-P organisms such as M. parvicella could be increased under low Food/ Microorganism (F/M) conditions. Furthermore, Tsai et al. (2003) showed that ammonia is available for the growth of M. parvicella. In this research, low F/M ratio (about 400 mg L 1 COD and 4000 mg L 1 MLSS) was calculated in influent, in addition, because of low pH values (7 ± 0.1) during aerobic period, the activity of nitrifying bacteria was strongly restrained (Jetten et al., 2001), as a result, there is enough ammonia to be utilized as nitrogen source for growth by M. parvicella in the system. Therefore, we proposed a hypothesis like this: since the environ-

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Fig. 4. Poly-P hydrolysis, PO4-P release in the settle/decant/idle zone as well as MLVSS/MLSS transition during long-term operation in sequencing batch reactor. The samples were taken from the solution at the end of aeration.

mental conditions were very fit to growth of poly-P organisms, biomass of poly-P organisms increased gradually and became dominant bacteria in the activated sludge system, and then P removal was mainly conducted by other polyP organisms such as M. parvicella in this research. Fig. 4 can provide the hypothesis strongly supports chemically. Although poly-P hydrolysis and PO4-P release in the settle/decant/idle zone were increased gradually at first 18 days, the contents were both at low levels, which implied that traditional PAOs existed but were in a subsidiary activity in this SBR systems, and P removal in this study were operated in both PAOs and other poly-P organisms at first 18 days. The lower poly-P hydrolysis and PO4-P release were observed in the settle/decant/idle zone after 18 days, suggesting a reduction in the PAOs activity in systems, however, the lower MLVSS/MLSS ratio implied that there was a higher level of poly-P in the sludge (Oehmen et al., 2005b). After 32-day transition period, steady low poly-P hydrolysis and PO4-P release as well as MLVSS/ MLSS ratio was very well explained by a population shift from PAOs to other poly-P organisms. Further investigations are necessary to explore the energy material which replaces PHA and to assess PAOs coupled with poly-P organisms activities in this study with the use of microbiological analytical techniques. 5. Conclusions The results of this study showed that P removal could be achieved in SBR with single-stage oxic process without PHA accumulation which was conventionally considered as key energy for P removal. The results also suggested that P was mainly removed in the form of poly-P, and about 207.235 mg phosphates removed by the discharge of richphosphorus sludge for each cycle in this study.

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