Environmental Technology Innovative anaerobic

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Innovative anaerobic/upflow sludge blanket filtration bioreactor for phosphorus removal from wastewater H. Khorsandia; H. Movahedyanb; B. Binab; H. Farrokhzadehb a Department of Environmental Health, Urmia University of Medical Sciences, Urmia, Iran b Department of Environmental Health, Isfahan University of Medical Sciences, Isfahan, Iran Online publication date: 03 May 2011

To cite this Article Khorsandi, H. , Movahedyan, H. , Bina, B. and Farrokhzadeh, H.(2011) 'Innovative anaerobic/upflow

sludge blanket filtration bioreactor for phosphorus removal from wastewater', Environmental Technology, 32: 5, 499 — 506 To link to this Article: DOI: 10.1080/09593330.2010.504235 URL: http://dx.doi.org/10.1080/09593330.2010.504235

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Environmental Technology Vol. 32, No. 5, April 2011, 499–506

Innovative anaerobic/upflow sludge blanket filtration bioreactor for phosphorus removal from wastewater H. Khorsandia*, H. Movahedyanb, B. Binab and H. Farrokhzadehb a

Department of Environmental Health, Urmia University of Medical Sciences, Urmia, Iran; bDepartment of Environmental Health, Isfahan University of Medical Sciences, Isfahan, Iran (Received 1 February 2010; Accepted 22 June 2010 )

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Downloaded By: [khorsandi, Hassan] At: 12:58 4 May 2011

10.1080/09593330.2010.504235

Phosphorus is the key element to remove from aquatic environments to limit the growth of aquatic plants and algae and, thus, to control eutrophication. Because the upflow sludge blanket filtratio’ (USBF) process, without addition of metal salts, entails low efficiency for phosphorus removal, we added an anaerobic reactor to the USBF bioreactor in order to promote the simultaneous removal of phosphorus and nitrogen from wastewater. The results revealed that the anaerobic/USBF bioreactor had a phosphorus removal efficiency up to 86%, with a sludge retention time (SRT) of 10 days, a hydraulic retention time (HRT) of 24 hours and an optimum COD/N/P ratio of 100/5/1. This ratio also improved the compaction quality of the sludge blanket in the USBF clarifier. The average specific phosphate uptake rate in the aerobic zone and the average specific phosphate release rate in the anaerobic reactor were 0.014 mg PO 4P removed/(g VSS·min) and 0.0525 mg PO4-P released/(g VSS·min), respectively. Secondary phosphorus release in the USBF clarifier was heightened with increasing HRT. Hence, the optimum total HRT can be selected between 16 and 24 hours based on effluent quality. Effluent phosphorus of about 1 mg/L was provided for wastewater with the COD/N/P ratio of 100/5/1 at the sludge age of 10 days and total HRT of 16 hours. This study illustrated that the anaerobic/USBF bioreactor at the optimum operational conditions can be an effective process for phosphorus removal from municipal wastewater. Keywords: municipal wastewater; phosphorus; biological removal; anaerobic; upflow sludge blanket filtration

Introduction Phosphorus and nitrogen removal from wastewater is an effective approach for prevention of eutrophication in water bodies. The problems caused by eutrophication have frequently been discussed in the literature and vary from a decrease in the aesthetics and depletion of the quality of the affected receiving water bodies to an increase in treatment costs [1–3]. Biological removal of phosphorus and nitrogen is usually integrated in wastewater treatment systems whenever treated effluent is to be discharged to sensitive receiving water or is to be exploited for reuse [3–5]. There are various configurations in combined nitrogen and phosphorus removal processes, based on influent characteristics, effluent limits and desired operational conditions. All of these processes consist of the same basic anaerobic/anoxic/aerobic components to achieve nitrification/denitrification and enhanced biological phosphorus uptake. In these processes, addition of chemical compounds and filtration of final effluent through the sand or other media are required for the removal of particulate matter when low nitrogen and phosphorus in the effluent are desired [4,5]. *Corresponding author. Email: [email protected] ISSN 0959-3330 print/ISSN 1479-487X online © 2011 Taylor & Francis DOI: 10.1080/09593330.2010.504235 http://www.informaworld.com

The upflow sludge blanket filtration (USBF) process is a novel configuration that incorporates an anoxic selector zone, an aeration unit and an upflow sludge blanket filtration clarifier in one integrated bioreactor [6]. In the USBF plant, wastewater enters the anoxic compartment where it mixes with activated sludge recycled from the bottom of the clarifier. The mixed liquor eventually underflows into the aerobic compartment. After aeration, a stream of the mixed liquor enters the bottom of a prism or cone-shaped clarifier and, as it rises, upward velocity decreases until the flocs of cells become stationary. Then, sludge flocs are separated from the liquid by upflow sludge blanket filtration, and clear effluent overflows into a collection trough and is discharged from the system [6]. The enhanced biological phosphorus removal (EBPR) process works by providing an anaerobic zone with an ample supply of readily biodegradable carbonaceous oxygen demand (rbCOD). Organic matter in the anaerobic zone is fermented to create volatile fatty acids (VFAs) as carbon sources for polyphosphateaccumulating organisms (PAOs). The PAOs can take up the VFAs from the bulk liquid and store them internally


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by linking the VFAs together to form complex longchain carbon molecules of poly-β-hydroxyalkanoates (PHAs). Phosphate-accumulating organisms obtain energy for producing intracellular PHA storage products by metabolizing stored polyphosphate, and thereby release phosphorus in bulk liquid during this phase in every EBPR system. The net increase in the amount of dissolved phosphate across the anaerobic phase of the process corresponds with this process [3,4,7]. In the aerobic phase of the process, PAOs are able to store more phosphate than is released under anaerobic conditions because considerably more energy is produced by aerobic oxidation of the stored PHAs than is used to store them under anaerobic conditions. Finally, net phosphorus removal from the wastewater is achieved through the removal of waste activated sludge containing high polyphosphate content [3,4,7]. Although most of the phosphorus removal is often achieved through anaerobic–aerobic cycling in the EBPR process, the anaerobic–anoxic operation also performs phosphorus removal as a result of the ability of denitrifying PAOs (DPAOs) to use nitrate or nitrite instead of oxygen as electron acceptors. Therefore, DPAOs perform phosphorus uptake and denitrification simultaneously [8,9]. Conventional activated sludge microorganisms contain 1.5–2.0% phosphorus [4]. This can give a removal of about 15–25% of the phosphorus in many municipal wastewaters [3]. Enhanced biological phosphorus removal processes are designed to culture communities of PAOs that have the ability to store intracellular phosphorus as high as 20–30% by dry weight [4]. Subsequent phosphorus removal efficiency can be 80–90%. When operated successfully, the EBPR process is a relatively inexpensive and environmentally sustainable option for P removal; however, the stability and reliability of EBPR can be a problem. Successful operation of the EBPR process depends on numerous process operational factors. Process upsets and the deterioration of P removal in the EBPR plants can be explained by such disturbances as the presence of nitrate in the anaerobic zone, potassium and/or magnesium limitation, overaeration and microbial competition of glycogen accumulating organisms (GAOs) with PAOs [2,10]. (GAOs) are a competing group of bacteria, and they use glycogen as the energy source for VFA uptake and storage, and obtain the needed reducing power through glycolysis. Glycogen-accumulating organisms have similar features to PAOs except they do not accumulate excess phosphorus and they tend to produce poly-hydroxyvalerate from VFAs. Consequently, they lack EBPR ability [11]. The process design considerations for BPR processes include wastewater characteristics, anaerobic contact

time and solids retention time (SRT). Process control is also necessary for increasing BPR efficiency [3–5]. The effects and importance of the COD/P ratio on EBPR performance in this other configuration have been presented and discussed by several researchers [5,12– 15]. The results of all studies agree that the minimum effluent P concentrations will be obtained when EBPR systems are operating under P-limiting conditions (when the COD/P ratio is high). Barnard et al. [12] established a relationship between the influent COD/P ratio and the effluent P concentration, and suggested that a COD/P ratio greater than 40/1 is usually needed to obtain an effluent P concentration less than 1.0 mg/L. All biological nutrient removal (BNR) processes must be either phosphorus limited or COD limited. Tay et al. [16] examined the effects of COD/N/P ratio on nitrogen and phosphorus removal in a single upflow fixed-bed filter provided with anaerobic, anoxic and aerobic conditions. They revealed that phosphorus removal efficiency was affected more by its own concentration than by COD and N concentrations, whereas nitrogen removal efficiency was unaffected by different phosphorus concentrations. At the COD/N/P ratio of 300/5/1, both nitrogen and phosphorus were effectively removed using the filter, with removal efficiencies of 87% and 76%, respectively. For biological phosphorus removal processes, hydraulic retention time (HRT) is an important factor for reliable phosphorus release in the anaerobic reactor and phosphorus uptake in the aerobic zone. Decreasing HRT in the suitable range increases both phosphorus and nitrogen removal efficiencies because, as HRT decreases, the food-to-microorganism loading ratio increases. Therefore, HRT should be considered an important factor in achieving better nutrient removal efficiency in every process. It was included in reference [17]. Stephen and Stensel [15] found that secondary phosphorus release occurred for anaerobic contact times in excess of three hours. Mahvi et al. [18] indicated that the efficiency of a single-stage USBF for phosphorus removal was 55% at the aeration time of six hours. However, effective removal of phosphorus can be achieved in all systems by adding metal salt to the aeration zone immediately prior to the mixed liquor entering the clarifier [3–5,6]. Because of the high costs of metal salts and the undesired efficiency of the single-stage USBF for phosphorous removal, we developed the novel anaerobic/USBF system in order to promote the simultaneous removal of phosphorous and nitrogen from wastewaters. The objective of our recent paper is to evaluate the ability of this novel system to accomplish EBPR. It is worth noting that, prior to this study, no trial had been done for investigating the efficiency of this process in wastewater treatment.



by a Hailea air compressor, with a maximum discharge of 90 L/min, and injected via three parallel tube diffusers. The airflow rate was regulated with three manual valves to supply the dissolved oxygen demand (DO) concentration of 4 ± 0.5 mg/L in the mixed liquor of the aerobic zone. The mixing of anaerobic and anoxic reactor contents was carried out by a mixer with a speed of 32 rpm.

Materials and methods Experimental set-up The experiments were performed using a laboratoryscale anaerobic/USBF bioreactor. A sketch of this labscale reactor is shown in Figure 1 and some key parameters are listed in Table 1. An anaerobic/aerobic configuration was installed for performance of the EBPR. The PAO- and phosphorusrich return activated sludge (RAS) was recycled to the anaerobic zone to build up the population of PAOs in the system and to be reconditioned for another EBPR cycle. In this process, the anoxic zone was built for nitrate reduction by denitrifiers and phosphorus uptake by denitrifying polyphosphate accumulating organisms (DPAOs). The functions of the aeration zone were nitrification and phosphorus luxury uptake. The recycling sludge from the USBF clarifier was directed to the anoxic zone by a submersible pump. The rate of return of RAS from the clarifier was controlled and monitored by a full automatic timer, which controlled the flow rate and time of sludge return. Mixed liquor from the anoxic zone was recycled to the anaerobic reactor by an Etatron pump for EBPR implementation. The RAS and anoxic recirculation rates were typically four and two times the influent flow rate, respectively. The combined anaerobic/USBF bioreactor was placed into a water bath equipped with aquarium heaters and a thermocouple in order to operate at the constant temperature of 28 ± 1 °C. Required air for aeration and mixing in the aerobic zone was supplied Figure 1.

Schematic diagram of the lab-scale anaerobic/USBF combined bioreactor/.

Figure 1. Table 1.

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Operating procedure The synthetic wastewater containing glucose as the main organic source, NH4HCO3 as nitrogen (N) source, and KH2PO4 and K2HPO4 as phosphorus (P) sources was introduced to the system during operation. The synthetic wastewater characteristics are listed in Table 2. Trace elements included FeCl3·6H2O 1.5 g/L, H3BO3 0.15 g/L, CuSO4·5H2O 0.03 g/L, KI 0.03 g/L, MnCl2·4H2O 0.12 g/L, NaMoO4·2H2O 0.06g/L, ZnSO4·H2O 0.12 g/L, CoCl2·6H2O 0.15 g/L. Sludge obtained from the Isfahan municipal wastewater treatment plant was used as a seed. The composition of ingredients in the synthetic wastewater was determined on the basis of desired chemical oxygen demand (COD) concentration, COD/N and COD/P ratios. In order to evaluate the effects of COD/N/P ratios on the efficiency of the system and to reach the optimum ratio, COD/N and COD/ P ratios ranged from 20 to 6 and from 100 to 30 respectively, based on the constant COD concentration of 750 mg/L. Then, the concentration range of NH4-N and PO4P were 37.5–125 mg/L and 7.5–25 mg/L, respectively.

Schematic diagram of the lab-scale anaerobic/USBF combined bioreactor. Operational data for the lab-scale anaerobic/USBF combined bioreactor.

Parameter Volume (L) Flow rate (L/d) HRT (h) SRT (day) Average MLSS (mg/L) at the optimum ratio Average MLVSS (mg/L) at the optimum ratio

Anaerobic reactor 3

Anoxic reactor 6

Aerobic reactor 9

USBF clarifier 2

10–60 1.2–7.2 – 2605 2115

2.4–14.4 – 3290 2575

3.6–21.6 10–30 3735 2865

0.8–4.8 – 6200 –

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Table 2. study.

H. Khorsandi et al. Composition of synthetic wastewater used in this

Chemicals

Concentrations

C6H12O6. NH4HCO3 KH2PO4 K2HPO4 MgSO4·7H2O CaCl2·2H2O FeCl3·6H2O Trace elements

Variable as influent COD Variable as COD/N Variable as COD/N Variable as COD/N As Mg/P = 0.56/1 As Ca/P = 0.32/1 As Fe/COD = 0.5/100 0.5 mL/L

The COD/P ratio for some locations around the world was determined to be from 27 to 91 [12]. In order to demonstrate the ability of system to accomplish EBPR for wastewaters with high COD, influent COD varied from 750 to 2000 mg/L at the optimum COD/N/P ratio. The prepared wastewater was continuously pumped into the anaerobic/USBF bioreactor at the flow rate of 20 L/day under the aforementioned conditions. Using this flow rate resulted in an HRT of 3.6 h, 7.2 h and 10.8 h, in the anaerobic, anoxic and aerobic reactors, respectively. In the series of experiments assessing the influence of various HRT and COD/N/P ratios on system efficiency, an SRT of 25 days was maintained. At the optimum COD/N/P ratio, overall HRT was set variably from 12 to 48 hours in order to select the optimum HRT. Mass balance calculations were developed to determine the actual changes in phosphorus and other parameters in each reactor on the basis of influent, effluent and return flows [4]. At the end, in order to estimate the values of the investigated kinetic parameters, the continuous feeding of the treating system was interrupted for the optimum HRT, and the pilot plant was turned into a batch mode of operation by hydraulic isolation of each single tank. Instant addition of nitrate (about 30 mg NO3-N/L) and organic substrate (about 200 mg COD/L) took place directly in the anoxic compartment. At the same time, ammonia was added in excess amount (30 mg NH-N/L) in the aerobic tanks. Samples of mixed liquor were taken from each reactor at fixed time intervals and were immediately analysed. Kinetic parameters of phosphorus (uptake and release rate) were determined using anaerobic, aerobic and anaerobic batch steps. Sampling and analysis The system was monitored for about one month, allowing it to reach steady-state conditions, and then operated for seven months. At least three runs of steady-state

data were collected from each reactor during each phase of the experiments to characterize each of the data. Samples were collected from the influent, effluent and sampling port of each reactor. Temperature, dissolved oxygen (DO) and pH were measured daily in each reactor immediately before sampling. The DO measurements were carried out using a YSI 55 DO meter (YSI Company Inc., Yellow Spring, Ohio, USA) and a Schott pH meter model CG-824 (Schott UK Ltd., Stafford, UK) was used for pH analysis. The samples were analysed immediately after centrifugation. Soluble chemical oxygen demand (sCOD), ammonium (NH4N), nitrate (NO3-N), nitrite (NO2-N), soluble phosphorus (PO4-P), mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were analysed in accordance with Standard Methods [19]. Results Because the blanket stability in the USBF clarifier was affected by nitrogen concentration, the COD/N and COD/P ratios were decreased simultaneously in the influent wastewater. The COD/P ratios of 100, 80, 70, 60, 50, 40 and 30 were examined at a constant COD of 750 mg/L, COD/N ratios varying from 20 to 6, total HRT of 24 hours and SRT of 25 days in order to evaluate the influence of COD/N/P ratios on the system efficiency and to reach the optimum ratio. The average phosphate removal efficiency in the anaerobic/USBF bioreactor versus COD/P variations is shown to decrease with decreasing COD/P ratio (Figure 2). The results indicated that the combined anaerobic/USBF bioreactor has a phosphorus removal efficiency of 81.33% at the optimum COD/N/P ratio of 100/5/1. In addition to high phosphorus removal efficiency at this ratio, the compaction of the sludge blanket was better than that at other ratios. As expected, the average phosphorus removal efficiency was promoted to 86% at the SRT of 10 days (see Figure 3). Figure 4 shows that the aerobic phosphate removal rate has a strong correlation with the aerobic phosphate loading rate (R2 =0.9969). This result is in accordance with other studies [13,20,21]. On the other hand, the aerobic reactor efficiency for phosphorus removal decreased from 92.1% at the COD/P ratio of 100 to 49.7% at the COD/P ratio of 30. As a result, the anaerobic/USBF efficiency for phosphorus removal decreased with increasing aerobic phosphate loading rate. Of course, phosphorus and nitrogen loading rates were increased simultaneously in this study, thus leading to decrease in the phosphorus removal efficiency. Orthophosphate (PO4-P) is taken up from solution in the aerobic and anoxic zones, generally leading to very low remaining concentrations. Thus, determination of phosphorus removal efficiency in these reactors is Figure 3. 2.

The average phosphate removal efficiency versus SRT. COD/P (SRT = 25 days, HRT = 24 hours).



Figure 2. The average phosphate removal efficiency versus COD/P (SRT = 25 days, HRT = 24 hours).

Figure 3. SRT.

The average phosphate removal efficiency versus

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Figure 5. The average phosphorus removal efficiency in the reactors at the optimum ratio (SRT = 25 days, HRT = 24 hours).

figure and Table 1, the maximum phosphorus removal occurred in the aerobic zone with the average specific phosphate uptake rate of 0.014 mg PO4-P removed/(g VSS·min), and the average specific phosphate release rate was 0.0525 mg PO4-P released/(g VSS·min) in the anaerobic reactor. Based on Figure 6, these parameters increased to 0.029 mg PO4-P removed/(g VSS·min) and 0.069 mg PO4-P released/(g VSS·min) in the batch mode because of the decline in the possible interferences between the nitrogen and phosphorus removal processes. The aerobic phosphate removal rate had also a strong correlation with the anaerobic phosphate release rate (R2 = 0.8967). However, the ratio of anaerobic Prelease rate to aerobic P-removal rate decreased from 2.74, at the COD/P ratio of 100, to 2.45 at the COD/P ratio of 30 (Figure 7). The average phosphate removal efficiency versus the influent COD, nitrogen and phosphorus concentrations at the optimum COD/N/P ratio of 100/5/1 is shown in Figure 8. As can be seen from the figure, the phosphate removal efficiency increased with increasing influent Figure 6. 4. 5.

The profiles average of phosphate phosphorus the average uptake removal specific rateefficiency versus phosphorus theinaverage the uptake reactors phosphate rate at (SPUR) the loading optimum and specific rate ratio in the (SRT phosphorus aerobic = 25 days, reactor release HRT (SRT rate = 24 (SPRR) = 25 hours). days, in the HRT batch = 24mode. hours).

Figure 7.

The average aerobic phosphate uptake rate versus the average anaerobic phosphate release rate (SRT = 25 days, HRT = 24 hours).

Figure 4. The average phosphate uptake rate versus the average phosphate loading rate in the aerobic reactor (SRT = 25 days, HRT = 24 hours).

important. The results of the average phosphorus release in the anaerobic reactor and the average phosphorus removal in the anoxic and aerobic zones at the optimum ratio are shown in Figure 5. According to this

Figure 6. The profiles of the average specific phosphorus uptake rate (SPUR) and specific phosphorus release rate (SPRR) in the batch mode.

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Figure 7. The average aerobic phosphate uptake rate versus the average anaerobic phosphate release rate (SRT = 25 days, HRT = 24 hours). Downloaded By: [khorsandi, Hassan] At: 12:58 4 May 2011

Figure 9. The effect of total HRT on PO4-P, TN, SCOD and TSS removal efficiencies (SRT = 25 days).

effect of total HRT on the PO4-P, TN, sCOD and TSS removal efficiencies were examined in the lab-scale anaerobic/USBF. The results of these experiments showed that the optimum HRT was between 16 and 24 hours based on effluent quality. Figure 9.

Figure 8. The average phosphate removal efficiency versus influent COD at the optimum ratio (SRT = 25 days, HRT = 24 hours).

COD at the constant concentrations of nitrogen and phosphorus. A comparison of Figures 2 and 8 shows that the phosphate removal efficiency increased from 58.4% to 85.2% with increasing influent COD from 750 mg/L to 1250 mg/L at the influent nitrogen concentration of 62.5 mg/L and the influent phosphorus concentration of 12.5 mg/L. Also, the phosphate removal efficiency increased from 57.3% to 87.5% with increasing influent COD from 750 mg/L to 1500 mg/L at the influent nitrogen concentration of 75 mg/L and the influent phosphorus concentration of 15 mg/L. The effect of total HRT variations on the phosphorus removal efficiency of the anaerobic/USBF is shown in Figure 9. In order to select the optimum HRT, the Figure 8.

The average phosphate removal efficiency versus influent COD at the optimum ratio (SRT = 25 days, HRT = 24 hours).

The effect of total HRT on PO 4-P, TN, SCOD and TSS removal efficiencies (SRT = 25 days).

Discussion In the present study, the lowest effluent phosphorus concentration and the highest efficiency was achieved at the COD/P ratio of 100. Since the anaerobic/USBF configuration had been designed for simultaneous nitrogen and phosphorus removal, settling quality and secondary phosphorus release in the USBF clarifier were affected by N concentration. Laboratory experiments illustrated that secondary phosphorus release and rising sludge occurred in the USBF clarifier at the low COD/N/P ratios and/or high HRT. The difference in the phosphorus uptake rate in the main and batch systems confirmed interferences between the nitrogen and phosphorus removal processes. This result is consistent with the finding of Tay et al. [16]. Because the compaction of the sludge blanket was weak at a total HRT of less than 16 hours and more than 24 hours, the optimum total HRT can be selected between 16 and 24 hours (anaerobic contact time of 2.4–3.6 hours) for this configuration based on effluent quality. Other studies [15,17,21] substantiate this result. Under longer aerobic SRT, more oxidation of organic matter can be obtained, leading to a higher rate of nitrogen removal. However, longer aerobic SRT may adversely affect biological phosphorus removal as a



result of the secondary release of phosphorus due to an increase in endogenous respiration in the aerobic zone [4]. In addition, the final amount of phosphorus removed is proportional to the amount of stored biological phosphorus in wasted bacteria. Thus, there is an SRT conflict between the reactions of nitrogen and phosphate removal [4,22]. It has been reported that an SRT of 10–12 days is optimal for both N and P removal [23,24]. Our investigations about SRT were in agreement with the mentioned studies so that TN and TP removal efficiencies were determined to be 90.4% and 86%, respectively, at the sludge age of 10 days. When the sludge age increased to 25 days, these amounts were changed to 96.9% and 81.33%, respectively. The rbCOD is the primary to the formation of VFAs for the PAOs. The more acetate, the more cell growth, and, thus, the more phosphorus removal [4]. For this reason, BPR efficiency at the optimum ratio is increased with increasing influent COD. Effluent TSS was less than 10 mg/L at the optimum conditions. Thus, filtration of the final effluent through sand or other media is not required for the removal of particulate matter when low effluent phosphorus concentration is required. Consequently, the anaerobic/USBF bioreactor at the optimum conditions can be an effective technology for phosphorus removal from municipal wastewater. Conclusions A good separation and compaction of the sludge blanket in the USBF clarifier and efficient BPR in the anaerobic/ USBF bioreactor depends on wastewater characteristics, HRT, SRT and process control. Effluent phosphorus of about 1 mg/L was provided for wastewater with a COD/ N/P ratio of 100/5/1 at an SRT of 10 days and HRT of 16 hours. The anaerobic/USBF bioreactor at the optimum conditions can be an effective technology for phosphorus removal from municipal wastewater, but it is not suggested for wastewater containing a high TKN/COD ratio because of rising sludge in the USBF clarifier.

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Acknowledgement The authors would like to thank the research administration of Isfahan University of Medical Sciences for financial support to carry out this project under grant number 387154. The authors also appreciate the contribution of Mr. H. Farrokhzadeh for his assistance in constructing the lab-scale anaerobic/ USBF bioreactor.

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