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extended-idle regime
Accepted Manuscript Title: Effect of dissolved oxygen on biological phosphorus removal induced by aerobic/extended-idle regime Author: Hongbo Chen Dongbo Wang Xiaoming Li Qi Yang Kun Luo Guangming Zeng Maolin Tang Weiping Xiong Guojing Yang PII: DOI: Reference:
S1369-703X(14)00063-1 http://dx.doi.org/doi:10.1016/j.bej.2014.03.004 BEJ 5903
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
22-10-2013 14-1-2014 11-3-2014
Please cite this article as: H. Chen WangX. LiQ. YangK. LuoG. ZengM. TangW. XiongG. Yang Effect of dissolved oxygen on biological phosphorus removal induced by aerobic/extended-idle regime, Biochemical Engineering Journal (2014), http://dx.doi.org/10.1016/j.bej.2014.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of dissolved oxygen on biological phosphorus removal
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induced by aerobic/extended-idle regime
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Hongbo Chen, Dongbo Wang*, Xiaoming Li*, Qi Yang, Kun Luo, Guangming Zeng,
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Maolin Tang, Weiping Xiong, Guojing Yang
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College of Environmental Science and Engineering, Hunan University, Changsha
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410082, China
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Key Laboratory of Environmental Biology and Pollution Control (Hunan University),
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Ministry of Education, Changsha 410082, China
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* Corresponding authors. Address: College of Environmental Science and Engineering, Hunan University, Changsha 410082, China. Tel.: +86 731 88823967; fax: +86 731 88822829. E-mail address: [email protected] (D. Wang), [email protected] (X. Li). 1
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Abstract: Previous researches have suggested that biological phosphorus removal (BPR) from wastewater could be achieved by the aerobic/extended-idle (A/EI) regime.
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This study further investigated the effect of dissolved oxygen (DO) concentration on
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BPR induced by the A/EI regime. The experimental results show that 1 mg/L of DO
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in mixed liquor benefited the BPR performance while a higher DO level of 5 mg/L
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deteriorated BPR. Fluorescent in situ hybridization analysis demonstrated that the
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improvement at 1 mg/L of DO was due to the shift in bacterial population from
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glycogen accumulating organisms (GAOs) to polyphosphate accumulating organisms
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(PAOs). The mechanism studies revealed that DO level affected the transformations
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of polyhydroxyalkanoates and glycogen and the activities of exopolyphosphatase and
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polyphosphate kinase. In addition, the BPR performances between the A/EI regime
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and conventional anaerobic/oxic (A/O) process were compared. The results showed
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that the A/EI regime drove better BPR performance than the A/O process at both low
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and high DO levels. More PAO and less GAO abundances in the biomass might be
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the principal reason for the higher BPR efficiency in the A/EI regime. Furthermore, controlling DO at a low level of 0.5 mg/L to promote BPR was demonstrated in a real municipal wastewater. The the A/EI regime showed an excellent BPR performance at the low DO levels and had a better tolerance to oxygen-limited condition as compared
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to the A/O regime.
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Keywords: Dissolved oxygen concentration; Biological phosphorus removal;
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Aerobic processes; Biosynthesis; Enzyme activity; Waste-water treatment
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1.
Introduction Enhanced biological phosphorus removal (EBPR) is an effective and
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environmental technology to remove phosphorus (P) from wastewaters [1]. In EBPR
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systems, polyphosphate accumulating organisms (PAOs) are able to store P through
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sequential anaerobic-aerobic conditions, while another group of microorganisms
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known as glycogen accumulating organisms (GAOs) compete with PAOs for the
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available organic substrate without contributing to P removal [2]. Successful operation
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of the EBPR systems depends on numerous process operational factors. In some cases,
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process upsets and the deterioration of P removal in EBPR plants can be explained by
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disturbances such as the presence of nitrate in the anaerobic pools [3] whereas in still
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other cases, the microbial competition of GAOs with PAOs is verified to be the major
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reason [4].
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In recent decades, factors affecting the PAO-GAO competition have been the
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focus of many studies, and dissolved oxygen (DO) concentration has been reported to
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impact the PAO-GAO competition [5,6]. On one hand, PAOs need DO or nitrate to uptake P from wastewaters. On the other hand, the inflow of DO or nitrate into anaerobic pools will inhibit the anaerobic P release. Poor P removal performances and high quantities of tetrad-forming organisms (TFOs) were often observed at very high
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DO concentrations of 4.5-5.0 mg/L, while DO concentrations of approximately
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2.5-3.0 mg/L seemed to correlate with the dominance of PAOs [5]. Additionally, an
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increase in the abundance of Accumulibacter and a decrease in Competibacter were
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observed at a low DO level of 0.5 mg/L [7]. 3
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Recently, achieving higher P removal efficiency with less energy consumption
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has become a very urgent task for wastewater treatment plants (WWTPs). As aeration
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is a costly process and the DO level will affect the efficiency of the removal of
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contaminants (e.g., BPR), an important strategy to minimize energy consumption and
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enhance BPR in activated sludge processes is to control the DO level in the aerobic
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zones.
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It has been reported that BPR can be achieved in activated sludge systems
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without specific anaerobic pools if the idle period is extended to 210-450 min [8]. This
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operation was defined as the aerobic/extended-idle (A/EI) regime and the inducing
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mechanisms of P removal in this process were verified to be different from previous
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processes [9,10]. Compared to the conventional anaerobic/oxic (A/O) process, the A/EI
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regime has several advantages such as the higher tolerance of nitrate and greater BPR
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efficiency [11,12]. However, those previous studies described the general performance
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in terms of P removal without specifying the importance of DO effect on the
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competition of PAOs and GAOs and performance of BPR in the A/EI systems. Moreover, BPR induced by the A/EI regime with low DO levels, such as 1 mg/L, has never been reported. In view of the fact that the A/EI regime has no specific anaerobic period, the microbial metabolic pathway would be different from the A/O processes [9].
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Thus, the effect of DO concentration on BPR performance in the A/EI regime might
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also be distinct from that in the A/O process. The purposes of this work were: 1) to investigate the effect of DO concentration
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on BPR induced by the A/EI regime; 2) to compare the BPR performances between 4
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the A/EI and A/O regimes operated at different DO levels; 3) to evaluate the
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feasibility of controlling DO at low levels to improve BPR in the A/EI regime when
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receiving a real municipal wastewater.
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2. Materials and methods
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2.1. Sequencing batch reactor (SBR) operation at different DO concentrations
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Experiments were carried out in three lab-scale SBRs each with a working
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volume of 12 L. Seed sludge was inoculated into the three SBRs concurrently.
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Aeration and mixing were supplied through an air diffuser placed in the bottom of the
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SBRs, and DO concentrations in the three SBRs were monitored by WTW Multi 340i
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DO meters and were kept constant at 1, 3, and 5 mg/L, respectively. All SBRs were
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operated as the A/EI regime with 8 h per cycle. The cyclic profile is comprised of 210
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min aeration, 55 min settling, 5 min decanting, and 210 min idle periods. After the
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settling phase 8L of supernatant was discharged from all the reactors and replaced
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with 8 L of the synthetic medium at the beginning of the aerobic phase. 1.5 L of
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sludge mixtures from the SBRs were discharged daily at the end of aerobic zone but before settling, resulting in a sludge retention time (SRT) of 8 d. As comparison, three reproductive SBRs were operated as the A/O process. Each
cycle of the A/O SBRs consisted of 120 min anaerobic mix and 180 min aeration,
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followed by 55 min settling, 5 min decanting, and 120 min idle periods according to
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the literature [13]. 8 L supernatant was discharged from the A/O SBRs after settling
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period and was replaced with 8 L of the synthetic medium at the end of the idle period.
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The A/O SBRs were mixed with magnetic stirrers in the anaerobic period. During the 5
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aerobic stage, air was supplied into the A/O SBRs to control DO levels at 1, 3, and 5
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mg/L, respectively. The SRT in the SBRs was maintained at 8 d. The synthetic medium used as influent contained 15 mg/L PO43--P, 40 mg/ L
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NH4+-N, and 300 mg/L chemical oxygen demand (COD). Acetate was used as the
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sole carbon source because it was the most common volatile fatty acid (VFA) in
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domestic wastewaters [14]. The concentrations of the other nutrients in the synthetic
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medium were as follows: 0.005 g/L CaCl2, 0.01 g/L MgSO4·7H2O, and 0.5 mL/L
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trace element solution [2].
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2.2. Low DO concentration to improve BPR from municipal wastewater
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In order to reduce the air supply during the aerobic period, it is necessary to
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investigate the behavior of P transformation in the A/EI regime at low DO levels. The
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investigation was performed in four identical SBRs each with a working volume of 12
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L. Seed sludge was inoculated into the four SBRs, two of which were operated as the
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A/EI regime and the other two were operated as the A/O process. The two A/EI SBRs
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were operated at 1 and 0.5 mg/L of DO, and the average DO concentrations in the two A/O SBRs were 3 and 0.5 mg/L, respectively. The other operational conditions were the same as those described in Section 2.1 except that the SBRs in this study received real municipal wastewaters which were collected from the inlet well of a WWTP in
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Changsha, China. The main characteristics of the municipal wastewater are as follows:
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total phosphate (TP) 6.5-9.7 mg/L, soluble orthophosphate (SOP) 4.6-7.8 mg/L, total
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nitrogen (TN) 32.4-45.8 mg/L, ammonia nitrogen (NH4+-N) 23.9-36.2 mg/L, soluble
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COD 120-216 mg/L, pH 7.0-7.2. 6
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2.3. Analytical methods NH4+-N, NO2--N, NO3--N, SOP, TP, COD, volatile suspended solids (VSS), and
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total suspended solids (TSS) were measured using the standard methods for the
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examination of wastewaters [15]. Sludge glycogen, poly-3-hydroxybutyrate (PHB),
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poly-3-hydroxyvalerate (PHV), and poly-3-hydroxy-2-methylvalerate (PH2MV) were
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measured according to the literature [8]. The total polyhydroxyalkanoates (PHAs) were
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calculated as the sum of measured PHB, PHV, and PH2MV.
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4 , 6 -diamidino-2-phenylindole dihydrochloride (DAPI) staining was carried
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out to analyze intracellular poly-P granules [16]. Sludge samples used for staining were
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taken at the end of the aerobic period. Analysis of exopolyphosphatase (PPX) and
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polyphosphate kinase (PPK) activities was performed according to the literature [2].
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Fluorescence in situ hybridization (FISH) technique was the same as described in
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the literature [9]. The following oligonucleotide probes used for hybridization are listed
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in Supporting Information (SI) Table S1. 30 microscopic fields were analyzed for the
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hybridization of individual probes using a confocal scanning laser microscope (FV 500). FISH quantification was performed with image database software (VideoTesT Album3.0).
3. Results and discussion
3.1. Variations of DO and pH during one cycle in A/EI SBRs
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To verify the effect of DO concentration on pH variations, pH was not held
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constant in this study so that pH varied as they would in a full-scale wastewater
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treatment plant. DO and pH were monitored continuously and the cyclic profiles of 7
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pH and DO variations in A/EI SBRs are shown in Fig. 1. At low DO level, the pH
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increased sharply from 7.1 to 7.5 in the initial 45 min of the aerobic period and
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decreased to 7.2 at the end of aeration, then further decreased to the initial level of 7.1
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during the subsequent idle period. Similar variations were observed at moderate and
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high aeration, indicating little difference in pH variations.
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3.2. BPR performances in the A/EI SBRs at different DO concentrations
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It took about 21 d before effluent SOP concentration became stable in all SBRs,
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then the data were reported. Table 1 summarises the reactor performances of the three
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SBRs during the steady-state operation. From Table 1, it can be concluded that COD
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and total nitrogen (TN) removal efficiencies were insensitive to DO concentration.
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However, the data of effluent SOP concentrations and SOP removal efficiencies
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showed that BPR performance depended strongly on DO concentration. Along with
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the increase of DO concentration, BPR performance of the system gradually
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deteriorated. The highest SOP removal efficiency of 98.5% was obtained at low DO
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level of 1 mg/L. Moreover, both of the highest SOP uptake and release rates were also detected at low DO level.
Figure 2 shows the variations of SOP during one cycle in the SBRs. Aerobic SOP
uptake and idle SOP release characteristics were observed in all SBRs. In the SBR
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operated at 1 mg/L of DO, the SOP release during the aerobic and the extended-idle
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period reached 12 and 6.2 mg/L, respectively. In contrast, activated sludge exhibited
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less SOP release and uptake ability in the SBRs at high DO levels. The SOP uptake
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decreased from 18.6 to 12.8 mg/L along with DO concentration increased from 1 to 5 8
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mg/L. These results clearly showed that better BPR performance could be achieved at
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low DO levels. The variations in activated sludge characteristics were studied for different DO
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concentrations (Fig. 3). It can be seen from Fig. 3 that DO concentration plays an
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important role in the evolution of sludge properties. At low DO level, activated sludge
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seemed to be granulated and there was a tendency to produce granular sludge. At
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moderate DO level (3 mg/L), though substances that surround cells were disappear,
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the size of activated sludge was still great. In contrast, sludge was scattered and
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decreased in size at high DO level of 5 mg/L, this might be due to the excessive
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aeration.
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DO influence on the activities of key enzymes relevant to SOP release and uptake
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was examined (Table 2). Poly-P is built up from ATP by PPK, and poly-P hydrolysis
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occurs either by the reverse PPK reaction leading to ATP formation from ADP or via
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hydrolysis by PPX [17]. As shown in Table 2, activated sludge in the SBR operated at 1
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mg/L of DO showed higher specific PPX and PPK activities, which was consistent with the observed higher SOP release and uptake (Fig. 2). The quantitative analysis of PAOs and GAOs in activated sludge was carried out
via FISH technology (Table 2). The results showed that the abundances of PAOs and
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GAOs respectively accounted for 32.5% and 12.1% of total biomass in the SBR
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operated at 1 mg/L of DO concentration, with PAOs 20.5% more than GAOs. In
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contrast, the differences between PAO and GAO abundances were relatively small in
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other two SBRs. 9
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3.3. Mechanism for low DO concentration driving high BPR performance It has been reported that pH plays an important role in BPR systems [18]. Higher
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pH was found to be more beneficial for PAOs due to its improvement to anaerobic
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phosphate release, substrate uptake ratio, and denitrification, and a clear microbial
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community shift from PAOs to GAOs along with the increase in the pH values was
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found [13,19]. The BPR performance in the A/EI process depended strongly on pH
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control because pH affected intracellular glycogen and PHAs transformations, which
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thereby influenced the microbial competition between PAOs and GAOs [11]. In this
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study, there was little difference among pH variations at different DO levels,
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suggesting that the different BPR performances were not the result of pH influence.
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Aerobic granular sludge has recently received growing attention by researchers
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and technology developers worldwide. Compared to conventional activated sludge
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flocs, the granules have a high sludge settle-ability, an excellent performance, and a
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small space demand. Aeration rate plays important roles in granule formation: on one
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hand, it imposes the hydrodynamic conditions; on the other hand, it controls the oxygen transfer [20]. High aeration rates are generally applied for improving granule formation. However, it seemed that high DO level is not necessarily a decisive condition on the formation of aerobic granules [21]. As a meaningful example, aerobic
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granules have been formed in SBR at a DO level as low as 0.7 mg/L [22]. The
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variations in activated sludge characteristics show that low DO level was conducive
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to the cultivation of granular sludge, which might benefit to good BPR performance.
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It was reported that BPR was strongly dependent on SOP release and uptake, 10
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which were directly related to PPX and PPK activities, respectively [2]. Therefore, the
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variations of PPX and PPK activities would affect BPR performance. In Table 2, the
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highest activities of PPX and PPK were detected in the SBR at 1 mg/L of DO, and
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with the increase of DO concentration both PPX and PPK activities were found to be
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decreased. The results suggested that one reason for the low DO concentration
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improving BPR performance in the A/EI regime was ascribed to the enhancement of
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PPX and PPK activities.
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Besides, it is reported that BPR is closely related to intracellular PHAs and
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glycogen transformations [23]. In view of the fact that the transformations of poly-P,
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PHAs, and glycogen constituted the microbial metabolism of BPR systems, the
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release and uptake of SOP would influence the transformations of intracellular
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glycogen and PHAs [24]. As shown in Fig. 4, in the AEI SBRs, acetate was rapidly
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depleted accompanied by obvious SOP release and intracellular PHAs accumulation
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during aerobic period. At the same time, glycogen was decreased slightly. After most
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of the acetate had been consumed and SOP release reached the peak value at 45 min, considerable PHAs degradation and SOP uptake as well as obvious glycogen synthesis were observed concurrently. However, the SBR at low DO level showed higher SOP release/uptake and PHAs synthesis/degradation but lower glycogen variations. PHAs and glycogen transformations were found to be associated with PAO and
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GAO activities, with high glycogen accumulation indicating the activated GAO
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metabolism [23]. Therefore, the increase of glycogen accumulation measured at high 11
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DO level indicated a population shift toward GAOs at high DO level, which was
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consistent with the result reported in the literature [25]. Moreover, higher SOP release
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(6.17 mg/L) and less glycogen degradation (0.27 mmol-C/g-VSS) was measured
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during the idle period in the SBR operated at low DO concentration. These results
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indicated that the energy for bacterial maintenance during the idle period was mainly
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provided by poly-P hydrolysis at low DO level but was provided by both poly-P
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hydrolysis and glycogen degradation at high DO concentration. The intracellular
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biochemical transformations at low DO level enhanced the role of poly-P participating
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in PAO metabolism and correlated well with PAO metabolism, which provided PAOs
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advantages over GAOs.
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The stoichiometries including P release (Prel), PHAs synthesis (PHAssyn),
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glycogen degradation (Glydeg) and the percentages of PHB, PHV and PH2MV in total
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PHAs in the aerobic period of this study and those reported by previous researches
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were compared in Table 3. In the aerobic period, the SBRs had almost the same level
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of PHB and PH2MV accumulation, but the PHV synthesis at low DO level was lower. This revealed that higher PHAs synthesis at low DO level was due to the increase of PHB and PH2MV but not PHV. Recent literatures reported that the energy for acetate uptake by GAOs was mainly from glycogen degradation, and when fed with acetate
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GAOs tended to produce more PHV than PAOs because of the partial conversion of
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pyruvate to propionyl-CoA through the succinate-propionate pathway [2,23]. It seems
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that low DO level benefits acetate consumption of PAOs due to GAOs being
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inhibited. 12
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The microbial competition between GAOs and PAOs was commonly considered
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to be responsible for EBPR deterioration [13]. It has been reported that the composition
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of the bacterial population structure in BPR sludge shifted with DO concentration [7].
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The FISH analysis suggested that low DO level could provide PAOs advantage in the
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competition with GAOs, and GAOs tended to become stronger competitors with
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PAOs when DO level exceeded 5 mg/L. The results suggested that competition by
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GAOs with PAOs in the A/EI regime may be more problematic under over aeration.
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3.4. Comparison of BPR performances between the A/EI and A/O SBRs
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N and P removal performance in the A/O-SBRs was also monitored in long-term
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operation. Comparison of reactor performance between the A/EI and A/O SBRs after
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reaching steady-state operation is summarized in Table 1. Almost the same quantities
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of COD and TN were present in effluent at different DO concentrations in both the
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A/EI and A/O SBRs, indicating that COD and TN removal in the A/EI and A/O SBRs
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was insensitive to DO concentration. Besides, a slight higher TN removal efficiency
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was detected in the A/O SBRs at all investigated DO levels as compared to the A/EI SBRs. From Table 1, it could be also observed that the highest SOP removal efficiency was obtained at DO concentration of 3 mg/L in the A/O process, while 1 mg/L of DO caused the highest BPR performance in the A/EI regime. When at their
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optimum DO level, A/EI SBR had a lower effluent SOP concentration (0.23 ± 0.02 vs
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0.44 ± 0.06 mg/L), thus had a higher BPR efficiency than the A/O SBR (98.5 ± 1.3%
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vs 97.1 ± 1.6%). Moreover, the SOP removal efficiency respectively remained around
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83.5 ± 2.2% and 78.9 ± 1.9% in the A/EI and A/O SBRs at a high DO concentration 13
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of 5 mg/L, with the A/EI SBR 4.6% higher than the A/O SBR. The results clearly
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illustrated that though good SOP removal performance could be driven by both the
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A/EI and the A/O regimes, the A/EI regime drove better.
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Figure 5 shows the DAPI staining of the sludge samples collected from the A/O
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and A/EI SBRs operated at different DO levels. In Fig. 5, there were large proportions
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of cells stained blue in Fig. 5a and 5e, elucidating dominant poly-P containing cells in
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the A/EI SBR operated at low DO level and the A/O SBR operated at moderate DO
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level, which was in accordance with the BPR efficiency results. Moreover, more blue
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area in Fig. 5a, 5b and 5c was found as compared to Fig. 5d, 5e and 5f, respectively,
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which suggested that more PAOs were contained in the A/EI SBRs than those in the
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A/O SBRs, and was consistent with the higher SOP release and uptake rates measured
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in the A/EI SBRs.
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The bacterial populations in the A/EI and A/O SBRs were summarized in Table 2.
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It can be concluded from Table 2 that activated sludge in the A/EI SBRs contained
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more PAOs and less GAOs as compared to the A/O SBRs, which was consistent with the DAPI staining results in Fig. 5 and the BPR efficiency results shown in Table 1. Similar cyclic variations of pH and DO were obtained in the A/O SBRs at
different DO levels (Fig. S1, Supporting Information), suggesting that DO
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concentration has no obvious effect on pH variations in the A/O SBRs. The activities
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of PPX and PPK at 3 mg/L of DO were higher than those at DO concentrations of 1
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and 5 mg/L, which were also consistent with the high SOP release and uptake at 3
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mg/L of DO as compared to other DO levels (Table 2). The results indicated that the 14
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relationship between the BPR performance and the activities of PPX and PPK in the
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A/EI regime is similar to that in the A/O process. Moreover, the activities PPX and
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PPK in the A/O SBR were higher than those in the A/EI SBR when DO concentration
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was 3 mg/L, which was in accordance with the result that the SOP release in the A/O
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SBR at 3 mg/L of DO was twice or threefold higher than that in the A/EI process (Fig.
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4).
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Table 3 showed that the Prel/VFAup and Glydeg/VFAup in the anaerobic period of
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the A/O process were higher than those in the aerobic phase of the A/EI regime, which
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was consistent with the higher SOP release and glycogen consumption in the A/O
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SBRs as compared to the A/EI SBRs (Fig. 4). It can be seen from Table 3 that PHA
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fractions measured in the A/O SBRs at 1 and 5 mg/L of DO levels in this study
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showed some differences from previous investigations. A low PHB synthesis and a
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high PHV accumulation were monitored in the A/O SBRs at 1 and 5 mg/L of DO
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levels. Recent literatures reported that GAOs tended to produce more PHV than PAOs
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when fed with acetate [2,23]. This indicated that a high GAO abundance was contained in the A/O SBRs at 1 and 5 mg/L of DO, which was in accordance with the bacterial population results in Table 2 and explained the decrease in BPR performance (Table 1).
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The cyclic variations of acetate, SOP, PHAs and glycogen in the A/EI and A/O
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SBRs are presented in Fig. 4. Acetate consumption was completed in the anaerobic
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period of the A/O regime but was accomplished aerobically in the A/EI regime, which
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suggests a substantial difference in metabolic mechanism. Acetate uptake and PHAs 15
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accumulation required NADH and ATP, which were usually provided respectively by
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poly-P hydrolysis and glycogen degradation [26]. The TCA cycle seemed to supply
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both NADH and ATP for PHAs synthesize in the A/EI SBRs due to the negligible
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SOP release and glycogen consumption during PHAs accumulation, suggesting that
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some of the acetate consumed via TCA cycle. Therefore less PHAs was accumulated
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in A/EI SBRs than in the A/O SBRs. Moreover, much lower glycogen consumption
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was measured in the A/EI SBRs as compared to the A/O SBRs, indicating that low
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GAO abundance was contained the activated sludge cultured in the A/EI SBRs than in
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the A/O SBRs [23], which seems to be the principal reason for the A/EI regime driving
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higher BPR efficiency.
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3.5. BPR from municipal wastewater at low DO concentration
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Similar cyclic variations of pH and DO were obtained in the A/EI and A/O SBRs
te
d
327
(Fig. S2, Supporting Information), suggesting that DO level has no obvious effect on
329
pH variations in municipal wastewater. TN and SOP removal efficiencies were
330 331 332 333
Ac ce p
328
compared after the four municipal wastewater SBRs reached their stable state (Table 1). From Table 1, it can be seen that similar TN and SOP removal efficiencies were obtained in municipal wastewater than in synthetic wastewater in both the A/EI and A/O SBRs at the optimum DO levels. At a low DO level of 0.5 mg/L, TN and SOP
334
removal efficiencies increased to 89.4 ± 2.5% and 98.2 ± 1.4% in the A/EI SBR,
335
respectively, while SOP removal efficiency slightly decreased to 74.5 ± 1.6% and TN
336
removal was deteriorated in the A/O SBR. It can be concluded that BPR from
337
municipal wastewater was improved in the A/EI SBR at low DO level (0.5 mg/L), and 16
Page 16 of 35
338
DO control strategy for BPR enhancement from municipal wastewater in the A/EI
339
regime was feasible. In contrast, low DO concentration unfavorably influenced BPR
340
performance and deteriorated TN removal in the A/O process.
ip t
The cyclic profiles of SOP, acetate, PHAs and glycogen in the two A/EI SBRs
341
operated at DO levels of 0.5 and 1 mg/L are shown in Fig. 6. When BPR from
343
municipal wastewater was conducted in the A/EI SBRs, it was observed that the
344
decrease of DO concentration affected the transformations of intracellular PHAs and
345
glycogen. In the initial 45 min of the aerobic period, the low DO level (0.5 mg/L)
346
caused more PHB and more total PHAs. Besides, a higher SOP release and lower
347
glycogen consumption was also measured. During the consequent aerobic stage,
348
higher PHAs degradation resulted in greater SOP uptake and glycogen accumulation.
349
Because low aeration can efficient reduce energy consumption, the A/EI regime may
350
become a low energy consumption BPR process.
352 353 354 355 356
us
an
M
d
te
Moreover, the A/EI SBR drove higher BPR than the A/O SBR at low DO
Ac ce p
351
cr
342
concentration, suggesting that the A/EI regime had a better tolerance to oxygen-limited condition as compared with the A/O process. That is to say, the A/EI regime will have a great advantage over the conventional A/O processes at plateau sections and remote areas at high altitudes where oxygen is limited. 4. Conclusions 1 mg/L of DO in mixed liquor improved the BPR performance in the A/EI regime.
357 358
The improvement was due to the shift in bacterial population from GAOs to PAOs.
359
DO levels affected the transformations of PHAs and glycogen and the activities of 17
Page 17 of 35
PPX and PPK. In addition, the A/EI regime showed higher BPR performance than the
361
A/O process at low and high DO, and more PAO and less GAO abundances in the
362
biomass might be the principal reason for the higher BPR efficiency in the A/EI
363
regime. Furthermore, controlling DO at a low level of 0.5 mg/L to promote BPR was
364
demonstrated in a real municipal wastewater.
365
Acknowledgements
us
cr
ip t
360
This material is based upon work supported by the project of National Natural
366
Science Foundation of China (NSFC) (Nos. 51278175 and 51378188), National
368
Science Foundation of Jiangsu Province (BK2012253), Zhejiang Provincial Natural
369
Science Foundation of China (LQ12E08001), and Hunan Provincial Innovation
370
Foundation for Postgraduate (CX2014B137).
371
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process and molecular ecological studies, Water Res. 32 (4) (1998) 1035-1048.
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[28] W.T. Liu, K. Nakamura, T. Matsuo, T. Mino, Internal energy-based competition
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phosphorus removal reactors-effect of P/C feeding ratio, Water Res. 31 (6) (1997)
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[29] N. Yagci, N. Artan, E.U. Cokg or, C.W. Randall, D. Orhon, Metabolic model for
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organisms under anaerobic conditions, Biotechnol. Bioeng. 84 (3) (2003) 359-373.
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466
22
Page 22 of 35
Figure legends:
467
Fig. 1 Profiles of DO and pH variations during a typical cycle of the SBRs operated as
469
A/EI regimes at DO levels of 1, 3 and 5 mg/L.
470
Fig. 2 Variations of SOP release and uptake with DO concentration in the SBRs
471
operated as A/EI regimes at DO levels of 1, 3 and 5 mg/L.
472
Fig. 3 Micrographs of the activated sludge samples collected from the SBRs
473
operated as A/EI regimes at DO levels of 1 (a), 3 (b) and 5 mg/L (c). Bar=5 μm.
474
Fig. 4 Variations of SOP, acetate and intracellular PHAs as well as sludge glycogen
475
during a typical cycle of the SBRs operated A/EI (a, c and e) and A/O regimes (b, d
476
and f) at DO levels of 1 (a and b), 3 (c and d), 15 (e and f). The data reported are the
477
averages and their standard deviations in triplicate tests.
478
Fig. 5 Micrographs of DAPI staining of the activated sludge samples withdrawn at
479
the end of aeration in the A/EI (a, b and c) and A/O SBRs (b and d) at DO levels of 1,
480
3 and 5 mg/L, respectively.
482 483 484
cr
us
an
M
d
te
Ac ce p
481
ip t
468
Fig. 6 Variations of SOP, acetate and intracellular PHAs as well as sludge glycogen during a typical cycle of the SBRs operated as A/EI regimes (c and d) at DO levels of 0.5 and 1 mg/L, respectively.
23
Page 23 of 35
484
Table 1‐Summary of reactor performances of A/EI and A/O SBRs during steady‐state
485
operationa
real
5 A/O
1 3 5
Ac ce p
A/EI
A/O
486
a
3
real
0.5
real
D COD remo val efficie ncy (%) 93.9 ± 2.1 93.2 ± 1.7 93.5 ± 1.6 92.3 ± 1.4 92.4 ± 1.2 93.6 ± 1.4 83.3 ± 2.3 86.5 ± 3.1 81.7 ± 2.7 79.1 ± 2.5
ip t
TN remo val efficie ncy (%) 82.8 ± 1.6 81.3 ± 1.2 81.4 ± 1.3 84.7 ± 1.4 84.8 ± 2.2 84.6 ± 1.7 83.8 ± 2.1 89.4 ± 2.5 84.5 ± 1.4 73.6 ± 1.5
Efflue nt COD (mg/L )
cr
0.5
3
0.23 ± 0.02 0.62 ± 0.07 2.47 ± 0.14 1.71 ± 0.26 0.44 ± 0.06 3.16 ± 0.48 0.27 ± 0.07 0.11 ± 0.03 0.56 ± 0.08 1.53 ± 0.13
Efflue nt NO3‐‐ N (mg/L ) 1.66 ± 0.55 2.23 ± 0.75 2.39 ± 0.12 1.24 ± 0.63 1.43 ± 0.31 1.97 ± 0.54 1.49 ± 0.36 0.83 ± 0.12 1.82 ± 0.61 1.12 ± 0.52
an
1
synt hetic synt hetic synt hetic synt hetic synt hetic synt hetic real
M
1
Efflue Effluen nt t NO2‐‐ + NH4 ‐N N (mg/L) (mg/L ) 4.21 ± 1.01 ± 0.53 0.28 4.03 ± 1.24 ± 0.72 0.36 3.59 ± 1.48 ± 0.41 0.27 4.24 ± 0.64 ± 0.61 0.13 3.83 ± 0.81 ± 0.57 0.27 3.16 ± 1.03 ± 0.38 0.42 4.33 ± 0.97 ± 0.51 0.16 4.87 ± 0.53 ± 0.43 0.09 3.74 ± 0.97 ± 0.22 0.61 9.58 ± 0.36 ± 0.44 0.11
d
A/EI
SOP remov al efficie ncy (%) 98.5 ± 1.3 95.7 ± 1.8 83.5 ± 2.2 88.6 ± 1.4 97.1 ± 1.6 78.9 ± 1.9 95.5 ± 1.2 98.2 ± 1.4 90.7 ± 1.3 74.5 ± 1.6
te
Regi me
DO Influ Efflue (m ent nt g/L type SOP ) (mg/L )
CO
N
18.1 ± 0.72 20.3 ± 1.31 19.5 ± 1.28 23.2 ± 1.67 21.7 ± 1.42 19.3 ± 0.84 28.7 ± 2.62 23.1 ± 2.27 31.4 ± 2.86 35.9 ± 3.41
us
P
Results are the averages and their standard deviations in triplicate tests.
24
Page 24 of 35
Table 2‐ Activities of PPX and PPK and bacterial populations in A/EI and A/O SBRsa
A/O
a
synthetic
1
synthetic
3
synthetic
5
synthetic
1
real
0.5
real
3
real
0.5
real
23.7 ± 3.6 19.8 ± 2.9
14.3 ± 3.2
ip t
5
27.2 ± 5.1
16.5 ± 4.4
16.8 ± 3.6
cr
synthetic
25.3 ± 4.4 21.2 ± 3.5
14.7 ± 2.8 17.1 ± 3.4
29.6 ± 4.7
12.4 ± 3.1
33.7 ± 5.5
10.7 ± 2.6
23.3 ± 3.4 19.8 ± 3.1
13.2 ± 2.9 18.5 ± 2.7
The data reported are the averages and their standard deviations in triplicate tests. Percentage to all bacteria (EUBmix probe). c The unit is μmol pnitrophenol/(min∙mg protein). d The unit is μmol NADPH/(min∙mg protein). b
Ac ce p
488 489 490 491 492
3
us
A/EI
synthetic
Bacterial populationsb PAOmix (%) GAOmix (%) 32.5 ± 5.4 12.1 ± 3.3
an
A/O
1
Enzyme activities PPXc PPKd 0.029 ± 0.278 ± 0.003 0.005 0.014 ± 0.169 ± 0.004 0.003 0.008 ± 0.113 ± 0.003 0.004 0.031 ± 0.216 ± 0.003 0.005 0.037 ± 0.283 ± 0.005 0.007 0.014 ± 0.121 ± 0.003 0.004 0.024 ± 0.253 ± 0.002 0.006 0.027 ± 0.262 ± 0.003 0.005 0.034 ± 0.271 ± 0.005 0.007 0.009 ± 0.112 ± 0.003 0.008
M
A/EI
DO Influent (mg/L) type
d
Regime
te
487
25
Page 25 of 35
110 Aerobic
Influent
Setting
100
Extended idle
ip t
A/EI regime A/O process
cr
90
80 Influent
Anaerobic
us
SOP removal efficiency (%)
decanting
Setting
Aerobic
idle
decanting
1
3 DO level (mg/L)
an
70
M
497
5
498
Ac ce p
te
d
Graphical Abstract. This paper showed that the aerobic/extended-idle (A/EI) regime drove superior biological phosphorus removal than the conventional anaerobic/oxic (A/O) process at low DO level.
27
Page 26 of 35
498
Highlights: Low DO level was demonstrated to improve BPR in A/EI regime;
500
DO concentration could affect PPX and PPK activities;
501
The biomass cultured at low DO level contained more PAOs and less
502
GAOs;
503
A/EI regime drove better BPR than A/O process at both high and low
504
DO levels;
505
A/EI regime had a better tolerance to oxygen-limited condition than
506
A/O process.
an
us
cr
ip t
499
Ac ce p
te
d
M
507
28
Page 27 of 35
Figure 1
7.8
6 Aerobic period
Settling/decanting periods
Idle period pH (1 mg/L) pH (3 mg/L) pH (5 mg/L) DO (1 mg/L) DO (3 mg/L) DO (5 mg/L)
DO (mg/L)
ip t cr
7.4
120
180
240 300 Time (min)
360
an
60
420
480
2
0
ce pt
ed
M
0
us
7.2
7.0
4
Ac
pH
7.6
Page 28 of 35
Figure 2
20 1 mg/L DO 3 mg/L DO 5 mg/L DO
cr
ip t
12
8
us
SOP (mg/L)
16
an
4
0
SOP uptake
Idle SOP release
Ac
ce pt
ed
M
Aerobic SOP release
Page 29 of 35
Ac c
ep te
d
M
an
us
cr
ip t
Figure 3
Page 30 of 35
Ac c
ep te
d
M
an
us
cr
ip t
Figure 4
Page 31 of 35
Figure 5
3
2
Aerobic period
360
420
0 480
Idle period 6 SOP Acetate PHB 5 PHV PH2MV PHAs 4 Glycogen
15
10
3 2
5 1
0
0
e5
20
2
180 240 300 Time (min)
Aerobic period
420
10
1
60
120
0 480
Idle period
180 240 300 Time (min)
Ac
0
360
420
SOP (mg/L)
0
d5
50
4
40
3
2
1
0
6 5
30
20
4
f5
50
3 2 1 0 480
3
2
120
180
240 300 Time (min)
360
420
0 480
Idle period
Aerobic period
6
5 SOP Acetate 4 PHB PHV PH2MV 3 PHAs Glycogen 2
10
0
4
60
Anaerobic period
0
ed
15
0
360
SOP Acetate PHB PHV PH2MV PHAs Glycogen
5
0
120
ce pt
3
60
SOP (mg/L)
Acetate (mmol-C/L)
4
0
0
1
M
1
10
0
60
120
Anaerobic period
180
1
240 300 Time (min)
360
420
0 480
Idle period
Aerobic period
6 5 SOP Acetate PHB 4 PHV PH2MV 3 PHAs Glycogen
40
30
20
2
1
10
0
0
1
0
60
120
180
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
180 240 300 Time (min)
SOP (mg/L)
Acetate (mmol-C/L)
4
120
1
240 300 Time (min)
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
20
60
2
360
420
0 480
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
c5
0
20
ip t
0
30
SOP (mg/L)
0
2
5 SOP Acetate 4 PHB PHV PH2MV 3 PHAs Glycogen
cr
1
3
6
us
1
40
Idle period
Aerobic period
an
5
4
Anaerobic period
SOP (mg/L)
2
50
Acetate (mmol-C/L)
3
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
10
b5
Acetate (mmol-C/L)
15
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
2
6 SOP Acetate PHB 5 PHV PH2MV PHAs 4 Glycogen
Acetate (mmol-C/L)
3
Idle period
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
Acetate (mmol-C/L)
4
Aerobic period 20
SOP (mg/L)
a5
Page 32 of 35
Figure 6
4
6
60
120
180 240 300 Time (min)
Aerobic period
360
420
Idle period
6
4
4
0
2.5
60
120
180
Aerobic period
240 300 Time (min)
2.0
1.5
360
420
480
Idle period 0.5 mg/L DO 1 mg/L DO
1.0
60
120
180 240 300 Time (min)
360
420
0 480
ed
0
0.5
0.0
0
60
120
180
240 300 Time (min)
360
420
480
ce pt
0
2
5
M
2
10
Ac
PHAs (mmol-C/g-VSS)
0.5 mg/L DO 1 mg/L DO
15
0
480
0.5 mg/L DO 1 mg/L DO
20
us
0
Glycogen (mmol-C/g-VSS) PHB PHV and PH2MV (mmol-C/g-VSS)
0
Idle period
ip t
8
Aerobic period
cr
0.5 mg/L DO 1 mg/L DO
12 SOP (mg/L)
25
Idle period
an
Aerobic period
PHB PHV and PH2MV (mmol-C/g-VSS)
16
Page 33 of 35
ip t cr
This study a
A/EI
1 3 5 1 3 5
Smolders et al. [26]
A/O
Glydeg/VFAup (mmol-C/mmol-C)
PHAssyn/VFAup (mmol-C/mmol-C)
PHB (%)
PHV (%)
PH2MV (%)
0.048±0.003
0.42±0.04
71.6±2.31
11.3±1.25
17.1±1.84
0.066±0.009
0.23±0.03
68.7±2.53
20.8±1.38
10.5±0.76
0.089±0.007
0.14±0.01
52.5±1.37
35.0±1.21
12.5±0.97
0.17±0.03
0.33±0.05
23.7±1.16
45.6±2.05
30.7±2.21
0.30±0.06
0.13±0.02
0.49±0.07
78.4±2.34
14.4±1.09
7.2±0.44
0.20±0.05
0.19±0.03
0.33±0.04
54.5±2.06
46.6±2.15
1.9±0.12
0.48
0.50
1.33
90
10
0
0.086±0.006 0.071±0.007 0.064±0.005 0.21±0.03
ep te
A/O
Prel/VFAup (mmol-P/mmol-C)
4
an
DO (mg/L)
M
Regime
d
Study
us
Table 3-Comparisons of stoichiometries and percentages of PHB, PHV and PH2MV in total PHAs between the aerobic period and conventional anaerobic period (acetate as carbon source)
Ac c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Pereira et al. [27]
A/O
2
0.16
0.69
1.47
71.4
28.6
0
Liu et al. [28]
A/O
6
0.45
0.78
1.47
75.8
20.0
4.2
A/O
6
0.57
0.53
1.30
88
12
0
A/O
-
0.96
0.48
1.24
89
9
2
Lu et al. [30]
A/O
2
0.62
0.46
1.26
94
6
0
Winkler et al. [25]
A2/O
6.25
0.15
0.71
1.11
77.3
22.7
0
Filipe et al. [18] Yagci et al. [29]
Page 34 of 35
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ep te
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Page 35 of 35
S1369-703X(14)00063-1 http://dx.doi.org/doi:10.1016/j.bej.2014.03.004 BEJ 5903
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
22-10-2013 14-1-2014 11-3-2014
Please cite this article as: H. Chen WangX. LiQ. YangK. LuoG. ZengM. TangW. XiongG. Yang Effect of dissolved oxygen on biological phosphorus removal induced by aerobic/extended-idle regime, Biochemical Engineering Journal (2014), http://dx.doi.org/10.1016/j.bej.2014.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of dissolved oxygen on biological phosphorus removal
2
induced by aerobic/extended-idle regime
3
Hongbo Chen, Dongbo Wang*, Xiaoming Li*, Qi Yang, Kun Luo, Guangming Zeng,
4
Maolin Tang, Weiping Xiong, Guojing Yang
5
College of Environmental Science and Engineering, Hunan University, Changsha
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410082, China
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Key Laboratory of Environmental Biology and Pollution Control (Hunan University),
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Ministry of Education, Changsha 410082, China
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* Corresponding authors. Address: College of Environmental Science and Engineering, Hunan University, Changsha 410082, China. Tel.: +86 731 88823967; fax: +86 731 88822829. E-mail address: [email protected] (D. Wang), [email protected] (X. Li). 1
Page 1 of 35
9
Abstract: Previous researches have suggested that biological phosphorus removal (BPR) from wastewater could be achieved by the aerobic/extended-idle (A/EI) regime.
11
This study further investigated the effect of dissolved oxygen (DO) concentration on
12
BPR induced by the A/EI regime. The experimental results show that 1 mg/L of DO
13
in mixed liquor benefited the BPR performance while a higher DO level of 5 mg/L
14
deteriorated BPR. Fluorescent in situ hybridization analysis demonstrated that the
15
improvement at 1 mg/L of DO was due to the shift in bacterial population from
16
glycogen accumulating organisms (GAOs) to polyphosphate accumulating organisms
17
(PAOs). The mechanism studies revealed that DO level affected the transformations
18
of polyhydroxyalkanoates and glycogen and the activities of exopolyphosphatase and
19
polyphosphate kinase. In addition, the BPR performances between the A/EI regime
20
and conventional anaerobic/oxic (A/O) process were compared. The results showed
21
that the A/EI regime drove better BPR performance than the A/O process at both low
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and high DO levels. More PAO and less GAO abundances in the biomass might be
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the principal reason for the higher BPR efficiency in the A/EI regime. Furthermore, controlling DO at a low level of 0.5 mg/L to promote BPR was demonstrated in a real municipal wastewater. The the A/EI regime showed an excellent BPR performance at the low DO levels and had a better tolerance to oxygen-limited condition as compared
27
to the A/O regime.
28
Keywords: Dissolved oxygen concentration; Biological phosphorus removal;
29
Aerobic processes; Biosynthesis; Enzyme activity; Waste-water treatment
2
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1.
Introduction Enhanced biological phosphorus removal (EBPR) is an effective and
31
environmental technology to remove phosphorus (P) from wastewaters [1]. In EBPR
33
systems, polyphosphate accumulating organisms (PAOs) are able to store P through
34
sequential anaerobic-aerobic conditions, while another group of microorganisms
35
known as glycogen accumulating organisms (GAOs) compete with PAOs for the
36
available organic substrate without contributing to P removal [2]. Successful operation
37
of the EBPR systems depends on numerous process operational factors. In some cases,
38
process upsets and the deterioration of P removal in EBPR plants can be explained by
39
disturbances such as the presence of nitrate in the anaerobic pools [3] whereas in still
40
other cases, the microbial competition of GAOs with PAOs is verified to be the major
41
reason [4].
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In recent decades, factors affecting the PAO-GAO competition have been the
43
focus of many studies, and dissolved oxygen (DO) concentration has been reported to
44 45 46 47
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impact the PAO-GAO competition [5,6]. On one hand, PAOs need DO or nitrate to uptake P from wastewaters. On the other hand, the inflow of DO or nitrate into anaerobic pools will inhibit the anaerobic P release. Poor P removal performances and high quantities of tetrad-forming organisms (TFOs) were often observed at very high
48
DO concentrations of 4.5-5.0 mg/L, while DO concentrations of approximately
49
2.5-3.0 mg/L seemed to correlate with the dominance of PAOs [5]. Additionally, an
50
increase in the abundance of Accumulibacter and a decrease in Competibacter were
51
observed at a low DO level of 0.5 mg/L [7]. 3
Page 3 of 35
Recently, achieving higher P removal efficiency with less energy consumption
52
has become a very urgent task for wastewater treatment plants (WWTPs). As aeration
54
is a costly process and the DO level will affect the efficiency of the removal of
55
contaminants (e.g., BPR), an important strategy to minimize energy consumption and
56
enhance BPR in activated sludge processes is to control the DO level in the aerobic
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zones.
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It has been reported that BPR can be achieved in activated sludge systems
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without specific anaerobic pools if the idle period is extended to 210-450 min [8]. This
60
operation was defined as the aerobic/extended-idle (A/EI) regime and the inducing
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mechanisms of P removal in this process were verified to be different from previous
62
processes [9,10]. Compared to the conventional anaerobic/oxic (A/O) process, the A/EI
63
regime has several advantages such as the higher tolerance of nitrate and greater BPR
64
efficiency [11,12]. However, those previous studies described the general performance
65
in terms of P removal without specifying the importance of DO effect on the
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competition of PAOs and GAOs and performance of BPR in the A/EI systems. Moreover, BPR induced by the A/EI regime with low DO levels, such as 1 mg/L, has never been reported. In view of the fact that the A/EI regime has no specific anaerobic period, the microbial metabolic pathway would be different from the A/O processes [9].
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Thus, the effect of DO concentration on BPR performance in the A/EI regime might
71
also be distinct from that in the A/O process. The purposes of this work were: 1) to investigate the effect of DO concentration
72 73
on BPR induced by the A/EI regime; 2) to compare the BPR performances between 4
Page 4 of 35
the A/EI and A/O regimes operated at different DO levels; 3) to evaluate the
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feasibility of controlling DO at low levels to improve BPR in the A/EI regime when
76
receiving a real municipal wastewater.
77
2. Materials and methods
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2.1. Sequencing batch reactor (SBR) operation at different DO concentrations
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Experiments were carried out in three lab-scale SBRs each with a working
80
volume of 12 L. Seed sludge was inoculated into the three SBRs concurrently.
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Aeration and mixing were supplied through an air diffuser placed in the bottom of the
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SBRs, and DO concentrations in the three SBRs were monitored by WTW Multi 340i
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DO meters and were kept constant at 1, 3, and 5 mg/L, respectively. All SBRs were
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operated as the A/EI regime with 8 h per cycle. The cyclic profile is comprised of 210
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min aeration, 55 min settling, 5 min decanting, and 210 min idle periods. After the
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settling phase 8L of supernatant was discharged from all the reactors and replaced
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with 8 L of the synthetic medium at the beginning of the aerobic phase. 1.5 L of
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sludge mixtures from the SBRs were discharged daily at the end of aerobic zone but before settling, resulting in a sludge retention time (SRT) of 8 d. As comparison, three reproductive SBRs were operated as the A/O process. Each
cycle of the A/O SBRs consisted of 120 min anaerobic mix and 180 min aeration,
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followed by 55 min settling, 5 min decanting, and 120 min idle periods according to
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the literature [13]. 8 L supernatant was discharged from the A/O SBRs after settling
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period and was replaced with 8 L of the synthetic medium at the end of the idle period.
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The A/O SBRs were mixed with magnetic stirrers in the anaerobic period. During the 5
Page 5 of 35
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aerobic stage, air was supplied into the A/O SBRs to control DO levels at 1, 3, and 5
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mg/L, respectively. The SRT in the SBRs was maintained at 8 d. The synthetic medium used as influent contained 15 mg/L PO43--P, 40 mg/ L
98
NH4+-N, and 300 mg/L chemical oxygen demand (COD). Acetate was used as the
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sole carbon source because it was the most common volatile fatty acid (VFA) in
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domestic wastewaters [14]. The concentrations of the other nutrients in the synthetic
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medium were as follows: 0.005 g/L CaCl2, 0.01 g/L MgSO4·7H2O, and 0.5 mL/L
103
trace element solution [2].
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2.2. Low DO concentration to improve BPR from municipal wastewater
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In order to reduce the air supply during the aerobic period, it is necessary to
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investigate the behavior of P transformation in the A/EI regime at low DO levels. The
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investigation was performed in four identical SBRs each with a working volume of 12
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L. Seed sludge was inoculated into the four SBRs, two of which were operated as the
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A/EI regime and the other two were operated as the A/O process. The two A/EI SBRs
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were operated at 1 and 0.5 mg/L of DO, and the average DO concentrations in the two A/O SBRs were 3 and 0.5 mg/L, respectively. The other operational conditions were the same as those described in Section 2.1 except that the SBRs in this study received real municipal wastewaters which were collected from the inlet well of a WWTP in
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Changsha, China. The main characteristics of the municipal wastewater are as follows:
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total phosphate (TP) 6.5-9.7 mg/L, soluble orthophosphate (SOP) 4.6-7.8 mg/L, total
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nitrogen (TN) 32.4-45.8 mg/L, ammonia nitrogen (NH4+-N) 23.9-36.2 mg/L, soluble
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COD 120-216 mg/L, pH 7.0-7.2. 6
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2.3. Analytical methods NH4+-N, NO2--N, NO3--N, SOP, TP, COD, volatile suspended solids (VSS), and
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total suspended solids (TSS) were measured using the standard methods for the
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examination of wastewaters [15]. Sludge glycogen, poly-3-hydroxybutyrate (PHB),
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poly-3-hydroxyvalerate (PHV), and poly-3-hydroxy-2-methylvalerate (PH2MV) were
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measured according to the literature [8]. The total polyhydroxyalkanoates (PHAs) were
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calculated as the sum of measured PHB, PHV, and PH2MV.
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4 , 6 -diamidino-2-phenylindole dihydrochloride (DAPI) staining was carried
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out to analyze intracellular poly-P granules [16]. Sludge samples used for staining were
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taken at the end of the aerobic period. Analysis of exopolyphosphatase (PPX) and
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polyphosphate kinase (PPK) activities was performed according to the literature [2].
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Fluorescence in situ hybridization (FISH) technique was the same as described in
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the literature [9]. The following oligonucleotide probes used for hybridization are listed
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in Supporting Information (SI) Table S1. 30 microscopic fields were analyzed for the
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hybridization of individual probes using a confocal scanning laser microscope (FV 500). FISH quantification was performed with image database software (VideoTesT Album3.0).
3. Results and discussion
3.1. Variations of DO and pH during one cycle in A/EI SBRs
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To verify the effect of DO concentration on pH variations, pH was not held
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constant in this study so that pH varied as they would in a full-scale wastewater
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treatment plant. DO and pH were monitored continuously and the cyclic profiles of 7
Page 7 of 35
pH and DO variations in A/EI SBRs are shown in Fig. 1. At low DO level, the pH
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increased sharply from 7.1 to 7.5 in the initial 45 min of the aerobic period and
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decreased to 7.2 at the end of aeration, then further decreased to the initial level of 7.1
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during the subsequent idle period. Similar variations were observed at moderate and
144
high aeration, indicating little difference in pH variations.
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3.2. BPR performances in the A/EI SBRs at different DO concentrations
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It took about 21 d before effluent SOP concentration became stable in all SBRs,
146
then the data were reported. Table 1 summarises the reactor performances of the three
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SBRs during the steady-state operation. From Table 1, it can be concluded that COD
149
and total nitrogen (TN) removal efficiencies were insensitive to DO concentration.
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However, the data of effluent SOP concentrations and SOP removal efficiencies
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showed that BPR performance depended strongly on DO concentration. Along with
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the increase of DO concentration, BPR performance of the system gradually
153
deteriorated. The highest SOP removal efficiency of 98.5% was obtained at low DO
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level of 1 mg/L. Moreover, both of the highest SOP uptake and release rates were also detected at low DO level.
Figure 2 shows the variations of SOP during one cycle in the SBRs. Aerobic SOP
uptake and idle SOP release characteristics were observed in all SBRs. In the SBR
158
operated at 1 mg/L of DO, the SOP release during the aerobic and the extended-idle
159
period reached 12 and 6.2 mg/L, respectively. In contrast, activated sludge exhibited
160
less SOP release and uptake ability in the SBRs at high DO levels. The SOP uptake
161
decreased from 18.6 to 12.8 mg/L along with DO concentration increased from 1 to 5 8
Page 8 of 35
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mg/L. These results clearly showed that better BPR performance could be achieved at
163
low DO levels. The variations in activated sludge characteristics were studied for different DO
164
concentrations (Fig. 3). It can be seen from Fig. 3 that DO concentration plays an
166
important role in the evolution of sludge properties. At low DO level, activated sludge
167
seemed to be granulated and there was a tendency to produce granular sludge. At
168
moderate DO level (3 mg/L), though substances that surround cells were disappear,
169
the size of activated sludge was still great. In contrast, sludge was scattered and
170
decreased in size at high DO level of 5 mg/L, this might be due to the excessive
171
aeration.
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DO influence on the activities of key enzymes relevant to SOP release and uptake
172
was examined (Table 2). Poly-P is built up from ATP by PPK, and poly-P hydrolysis
174
occurs either by the reverse PPK reaction leading to ATP formation from ADP or via
175
hydrolysis by PPX [17]. As shown in Table 2, activated sludge in the SBR operated at 1
177 178 179
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mg/L of DO showed higher specific PPX and PPK activities, which was consistent with the observed higher SOP release and uptake (Fig. 2). The quantitative analysis of PAOs and GAOs in activated sludge was carried out
via FISH technology (Table 2). The results showed that the abundances of PAOs and
180
GAOs respectively accounted for 32.5% and 12.1% of total biomass in the SBR
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operated at 1 mg/L of DO concentration, with PAOs 20.5% more than GAOs. In
182
contrast, the differences between PAO and GAO abundances were relatively small in
183
other two SBRs. 9
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3.3. Mechanism for low DO concentration driving high BPR performance It has been reported that pH plays an important role in BPR systems [18]. Higher
185
pH was found to be more beneficial for PAOs due to its improvement to anaerobic
187
phosphate release, substrate uptake ratio, and denitrification, and a clear microbial
188
community shift from PAOs to GAOs along with the increase in the pH values was
189
found [13,19]. The BPR performance in the A/EI process depended strongly on pH
190
control because pH affected intracellular glycogen and PHAs transformations, which
191
thereby influenced the microbial competition between PAOs and GAOs [11]. In this
192
study, there was little difference among pH variations at different DO levels,
193
suggesting that the different BPR performances were not the result of pH influence.
194
Aerobic granular sludge has recently received growing attention by researchers
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and technology developers worldwide. Compared to conventional activated sludge
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flocs, the granules have a high sludge settle-ability, an excellent performance, and a
197
small space demand. Aeration rate plays important roles in granule formation: on one
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hand, it imposes the hydrodynamic conditions; on the other hand, it controls the oxygen transfer [20]. High aeration rates are generally applied for improving granule formation. However, it seemed that high DO level is not necessarily a decisive condition on the formation of aerobic granules [21]. As a meaningful example, aerobic
202
granules have been formed in SBR at a DO level as low as 0.7 mg/L [22]. The
203
variations in activated sludge characteristics show that low DO level was conducive
204
to the cultivation of granular sludge, which might benefit to good BPR performance.
205
It was reported that BPR was strongly dependent on SOP release and uptake, 10
Page 10 of 35
which were directly related to PPX and PPK activities, respectively [2]. Therefore, the
207
variations of PPX and PPK activities would affect BPR performance. In Table 2, the
208
highest activities of PPX and PPK were detected in the SBR at 1 mg/L of DO, and
209
with the increase of DO concentration both PPX and PPK activities were found to be
210
decreased. The results suggested that one reason for the low DO concentration
211
improving BPR performance in the A/EI regime was ascribed to the enhancement of
212
PPX and PPK activities.
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Besides, it is reported that BPR is closely related to intracellular PHAs and
214
glycogen transformations [23]. In view of the fact that the transformations of poly-P,
215
PHAs, and glycogen constituted the microbial metabolism of BPR systems, the
216
release and uptake of SOP would influence the transformations of intracellular
217
glycogen and PHAs [24]. As shown in Fig. 4, in the AEI SBRs, acetate was rapidly
218
depleted accompanied by obvious SOP release and intracellular PHAs accumulation
219
during aerobic period. At the same time, glycogen was decreased slightly. After most
221 222 223 224
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of the acetate had been consumed and SOP release reached the peak value at 45 min, considerable PHAs degradation and SOP uptake as well as obvious glycogen synthesis were observed concurrently. However, the SBR at low DO level showed higher SOP release/uptake and PHAs synthesis/degradation but lower glycogen variations. PHAs and glycogen transformations were found to be associated with PAO and
225 226
GAO activities, with high glycogen accumulation indicating the activated GAO
227
metabolism [23]. Therefore, the increase of glycogen accumulation measured at high 11
Page 11 of 35
DO level indicated a population shift toward GAOs at high DO level, which was
229
consistent with the result reported in the literature [25]. Moreover, higher SOP release
230
(6.17 mg/L) and less glycogen degradation (0.27 mmol-C/g-VSS) was measured
231
during the idle period in the SBR operated at low DO concentration. These results
232
indicated that the energy for bacterial maintenance during the idle period was mainly
233
provided by poly-P hydrolysis at low DO level but was provided by both poly-P
234
hydrolysis and glycogen degradation at high DO concentration. The intracellular
235
biochemical transformations at low DO level enhanced the role of poly-P participating
236
in PAO metabolism and correlated well with PAO metabolism, which provided PAOs
237
advantages over GAOs.
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The stoichiometries including P release (Prel), PHAs synthesis (PHAssyn),
238
glycogen degradation (Glydeg) and the percentages of PHB, PHV and PH2MV in total
240
PHAs in the aerobic period of this study and those reported by previous researches
241
were compared in Table 3. In the aerobic period, the SBRs had almost the same level
243 244 245
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of PHB and PH2MV accumulation, but the PHV synthesis at low DO level was lower. This revealed that higher PHAs synthesis at low DO level was due to the increase of PHB and PH2MV but not PHV. Recent literatures reported that the energy for acetate uptake by GAOs was mainly from glycogen degradation, and when fed with acetate
246
GAOs tended to produce more PHV than PAOs because of the partial conversion of
247
pyruvate to propionyl-CoA through the succinate-propionate pathway [2,23]. It seems
248
that low DO level benefits acetate consumption of PAOs due to GAOs being
249
inhibited. 12
Page 12 of 35
The microbial competition between GAOs and PAOs was commonly considered
250
to be responsible for EBPR deterioration [13]. It has been reported that the composition
252
of the bacterial population structure in BPR sludge shifted with DO concentration [7].
253
The FISH analysis suggested that low DO level could provide PAOs advantage in the
254
competition with GAOs, and GAOs tended to become stronger competitors with
255
PAOs when DO level exceeded 5 mg/L. The results suggested that competition by
256
GAOs with PAOs in the A/EI regime may be more problematic under over aeration.
257
3.4. Comparison of BPR performances between the A/EI and A/O SBRs
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N and P removal performance in the A/O-SBRs was also monitored in long-term
259
operation. Comparison of reactor performance between the A/EI and A/O SBRs after
260
reaching steady-state operation is summarized in Table 1. Almost the same quantities
261
of COD and TN were present in effluent at different DO concentrations in both the
262
A/EI and A/O SBRs, indicating that COD and TN removal in the A/EI and A/O SBRs
263
was insensitive to DO concentration. Besides, a slight higher TN removal efficiency
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was detected in the A/O SBRs at all investigated DO levels as compared to the A/EI SBRs. From Table 1, it could be also observed that the highest SOP removal efficiency was obtained at DO concentration of 3 mg/L in the A/O process, while 1 mg/L of DO caused the highest BPR performance in the A/EI regime. When at their
268
optimum DO level, A/EI SBR had a lower effluent SOP concentration (0.23 ± 0.02 vs
269
0.44 ± 0.06 mg/L), thus had a higher BPR efficiency than the A/O SBR (98.5 ± 1.3%
270
vs 97.1 ± 1.6%). Moreover, the SOP removal efficiency respectively remained around
271
83.5 ± 2.2% and 78.9 ± 1.9% in the A/EI and A/O SBRs at a high DO concentration 13
Page 13 of 35
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of 5 mg/L, with the A/EI SBR 4.6% higher than the A/O SBR. The results clearly
273
illustrated that though good SOP removal performance could be driven by both the
274
A/EI and the A/O regimes, the A/EI regime drove better.
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Figure 5 shows the DAPI staining of the sludge samples collected from the A/O
275
and A/EI SBRs operated at different DO levels. In Fig. 5, there were large proportions
277
of cells stained blue in Fig. 5a and 5e, elucidating dominant poly-P containing cells in
278
the A/EI SBR operated at low DO level and the A/O SBR operated at moderate DO
279
level, which was in accordance with the BPR efficiency results. Moreover, more blue
280
area in Fig. 5a, 5b and 5c was found as compared to Fig. 5d, 5e and 5f, respectively,
281
which suggested that more PAOs were contained in the A/EI SBRs than those in the
282
A/O SBRs, and was consistent with the higher SOP release and uptake rates measured
283
in the A/EI SBRs.
te
The bacterial populations in the A/EI and A/O SBRs were summarized in Table 2.
284
286 287 288 289
It can be concluded from Table 2 that activated sludge in the A/EI SBRs contained
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more PAOs and less GAOs as compared to the A/O SBRs, which was consistent with the DAPI staining results in Fig. 5 and the BPR efficiency results shown in Table 1. Similar cyclic variations of pH and DO were obtained in the A/O SBRs at
different DO levels (Fig. S1, Supporting Information), suggesting that DO
290
concentration has no obvious effect on pH variations in the A/O SBRs. The activities
291
of PPX and PPK at 3 mg/L of DO were higher than those at DO concentrations of 1
292
and 5 mg/L, which were also consistent with the high SOP release and uptake at 3
293
mg/L of DO as compared to other DO levels (Table 2). The results indicated that the 14
Page 14 of 35
relationship between the BPR performance and the activities of PPX and PPK in the
295
A/EI regime is similar to that in the A/O process. Moreover, the activities PPX and
296
PPK in the A/O SBR were higher than those in the A/EI SBR when DO concentration
297
was 3 mg/L, which was in accordance with the result that the SOP release in the A/O
298
SBR at 3 mg/L of DO was twice or threefold higher than that in the A/EI process (Fig.
299
4).
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Table 3 showed that the Prel/VFAup and Glydeg/VFAup in the anaerobic period of
300
the A/O process were higher than those in the aerobic phase of the A/EI regime, which
302
was consistent with the higher SOP release and glycogen consumption in the A/O
303
SBRs as compared to the A/EI SBRs (Fig. 4). It can be seen from Table 3 that PHA
304
fractions measured in the A/O SBRs at 1 and 5 mg/L of DO levels in this study
305
showed some differences from previous investigations. A low PHB synthesis and a
306
high PHV accumulation were monitored in the A/O SBRs at 1 and 5 mg/L of DO
307
levels. Recent literatures reported that GAOs tended to produce more PHV than PAOs
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an
301
when fed with acetate [2,23]. This indicated that a high GAO abundance was contained in the A/O SBRs at 1 and 5 mg/L of DO, which was in accordance with the bacterial population results in Table 2 and explained the decrease in BPR performance (Table 1).
312
The cyclic variations of acetate, SOP, PHAs and glycogen in the A/EI and A/O
313
SBRs are presented in Fig. 4. Acetate consumption was completed in the anaerobic
314
period of the A/O regime but was accomplished aerobically in the A/EI regime, which
315
suggests a substantial difference in metabolic mechanism. Acetate uptake and PHAs 15
Page 15 of 35
accumulation required NADH and ATP, which were usually provided respectively by
317
poly-P hydrolysis and glycogen degradation [26]. The TCA cycle seemed to supply
318
both NADH and ATP for PHAs synthesize in the A/EI SBRs due to the negligible
319
SOP release and glycogen consumption during PHAs accumulation, suggesting that
320
some of the acetate consumed via TCA cycle. Therefore less PHAs was accumulated
321
in A/EI SBRs than in the A/O SBRs. Moreover, much lower glycogen consumption
322
was measured in the A/EI SBRs as compared to the A/O SBRs, indicating that low
323
GAO abundance was contained the activated sludge cultured in the A/EI SBRs than in
324
the A/O SBRs [23], which seems to be the principal reason for the A/EI regime driving
325
higher BPR efficiency.
326
3.5. BPR from municipal wastewater at low DO concentration
M
an
us
cr
ip t
316
Similar cyclic variations of pH and DO were obtained in the A/EI and A/O SBRs
te
d
327
(Fig. S2, Supporting Information), suggesting that DO level has no obvious effect on
329
pH variations in municipal wastewater. TN and SOP removal efficiencies were
330 331 332 333
Ac ce p
328
compared after the four municipal wastewater SBRs reached their stable state (Table 1). From Table 1, it can be seen that similar TN and SOP removal efficiencies were obtained in municipal wastewater than in synthetic wastewater in both the A/EI and A/O SBRs at the optimum DO levels. At a low DO level of 0.5 mg/L, TN and SOP
334
removal efficiencies increased to 89.4 ± 2.5% and 98.2 ± 1.4% in the A/EI SBR,
335
respectively, while SOP removal efficiency slightly decreased to 74.5 ± 1.6% and TN
336
removal was deteriorated in the A/O SBR. It can be concluded that BPR from
337
municipal wastewater was improved in the A/EI SBR at low DO level (0.5 mg/L), and 16
Page 16 of 35
338
DO control strategy for BPR enhancement from municipal wastewater in the A/EI
339
regime was feasible. In contrast, low DO concentration unfavorably influenced BPR
340
performance and deteriorated TN removal in the A/O process.
ip t
The cyclic profiles of SOP, acetate, PHAs and glycogen in the two A/EI SBRs
341
operated at DO levels of 0.5 and 1 mg/L are shown in Fig. 6. When BPR from
343
municipal wastewater was conducted in the A/EI SBRs, it was observed that the
344
decrease of DO concentration affected the transformations of intracellular PHAs and
345
glycogen. In the initial 45 min of the aerobic period, the low DO level (0.5 mg/L)
346
caused more PHB and more total PHAs. Besides, a higher SOP release and lower
347
glycogen consumption was also measured. During the consequent aerobic stage,
348
higher PHAs degradation resulted in greater SOP uptake and glycogen accumulation.
349
Because low aeration can efficient reduce energy consumption, the A/EI regime may
350
become a low energy consumption BPR process.
352 353 354 355 356
us
an
M
d
te
Moreover, the A/EI SBR drove higher BPR than the A/O SBR at low DO
Ac ce p
351
cr
342
concentration, suggesting that the A/EI regime had a better tolerance to oxygen-limited condition as compared with the A/O process. That is to say, the A/EI regime will have a great advantage over the conventional A/O processes at plateau sections and remote areas at high altitudes where oxygen is limited. 4. Conclusions 1 mg/L of DO in mixed liquor improved the BPR performance in the A/EI regime.
357 358
The improvement was due to the shift in bacterial population from GAOs to PAOs.
359
DO levels affected the transformations of PHAs and glycogen and the activities of 17
Page 17 of 35
PPX and PPK. In addition, the A/EI regime showed higher BPR performance than the
361
A/O process at low and high DO, and more PAO and less GAO abundances in the
362
biomass might be the principal reason for the higher BPR efficiency in the A/EI
363
regime. Furthermore, controlling DO at a low level of 0.5 mg/L to promote BPR was
364
demonstrated in a real municipal wastewater.
365
Acknowledgements
us
cr
ip t
360
This material is based upon work supported by the project of National Natural
366
Science Foundation of China (NSFC) (Nos. 51278175 and 51378188), National
368
Science Foundation of Jiangsu Province (BK2012253), Zhejiang Provincial Natural
369
Science Foundation of China (LQ12E08001), and Hunan Provincial Innovation
370
Foundation for Postgraduate (CX2014B137).
371
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process and molecular ecological studies, Water Res. 32 (4) (1998) 1035-1048.
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[28] W.T. Liu, K. Nakamura, T. Matsuo, T. Mino, Internal energy-based competition
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phosphorus removal reactors-effect of P/C feeding ratio, Water Res. 31 (6) (1997)
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[29] N. Yagci, N. Artan, E.U. Cokg or, C.W. Randall, D. Orhon, Metabolic model for
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organisms under anaerobic conditions, Biotechnol. Bioeng. 84 (3) (2003) 359-373.
[30] H. Lu, A. Oehmen, B. Virdis, J. Keller, Z.G. Yuan, Obtaining highly enriched cultures of Candidatus Accumulibacter phosphatis through alternating carbon sources, Water Res. 40 (20) (2006) 3838-3848.
466
22
Page 22 of 35
Figure legends:
467
Fig. 1 Profiles of DO and pH variations during a typical cycle of the SBRs operated as
469
A/EI regimes at DO levels of 1, 3 and 5 mg/L.
470
Fig. 2 Variations of SOP release and uptake with DO concentration in the SBRs
471
operated as A/EI regimes at DO levels of 1, 3 and 5 mg/L.
472
Fig. 3 Micrographs of the activated sludge samples collected from the SBRs
473
operated as A/EI regimes at DO levels of 1 (a), 3 (b) and 5 mg/L (c). Bar=5 μm.
474
Fig. 4 Variations of SOP, acetate and intracellular PHAs as well as sludge glycogen
475
during a typical cycle of the SBRs operated A/EI (a, c and e) and A/O regimes (b, d
476
and f) at DO levels of 1 (a and b), 3 (c and d), 15 (e and f). The data reported are the
477
averages and their standard deviations in triplicate tests.
478
Fig. 5 Micrographs of DAPI staining of the activated sludge samples withdrawn at
479
the end of aeration in the A/EI (a, b and c) and A/O SBRs (b and d) at DO levels of 1,
480
3 and 5 mg/L, respectively.
482 483 484
cr
us
an
M
d
te
Ac ce p
481
ip t
468
Fig. 6 Variations of SOP, acetate and intracellular PHAs as well as sludge glycogen during a typical cycle of the SBRs operated as A/EI regimes (c and d) at DO levels of 0.5 and 1 mg/L, respectively.
23
Page 23 of 35
484
Table 1‐Summary of reactor performances of A/EI and A/O SBRs during steady‐state
485
operationa
real
5 A/O
1 3 5
Ac ce p
A/EI
A/O
486
a
3
real
0.5
real
D COD remo val efficie ncy (%) 93.9 ± 2.1 93.2 ± 1.7 93.5 ± 1.6 92.3 ± 1.4 92.4 ± 1.2 93.6 ± 1.4 83.3 ± 2.3 86.5 ± 3.1 81.7 ± 2.7 79.1 ± 2.5
ip t
TN remo val efficie ncy (%) 82.8 ± 1.6 81.3 ± 1.2 81.4 ± 1.3 84.7 ± 1.4 84.8 ± 2.2 84.6 ± 1.7 83.8 ± 2.1 89.4 ± 2.5 84.5 ± 1.4 73.6 ± 1.5
Efflue nt COD (mg/L )
cr
0.5
3
0.23 ± 0.02 0.62 ± 0.07 2.47 ± 0.14 1.71 ± 0.26 0.44 ± 0.06 3.16 ± 0.48 0.27 ± 0.07 0.11 ± 0.03 0.56 ± 0.08 1.53 ± 0.13
Efflue nt NO3‐‐ N (mg/L ) 1.66 ± 0.55 2.23 ± 0.75 2.39 ± 0.12 1.24 ± 0.63 1.43 ± 0.31 1.97 ± 0.54 1.49 ± 0.36 0.83 ± 0.12 1.82 ± 0.61 1.12 ± 0.52
an
1
synt hetic synt hetic synt hetic synt hetic synt hetic synt hetic real
M
1
Efflue Effluen nt t NO2‐‐ + NH4 ‐N N (mg/L) (mg/L ) 4.21 ± 1.01 ± 0.53 0.28 4.03 ± 1.24 ± 0.72 0.36 3.59 ± 1.48 ± 0.41 0.27 4.24 ± 0.64 ± 0.61 0.13 3.83 ± 0.81 ± 0.57 0.27 3.16 ± 1.03 ± 0.38 0.42 4.33 ± 0.97 ± 0.51 0.16 4.87 ± 0.53 ± 0.43 0.09 3.74 ± 0.97 ± 0.22 0.61 9.58 ± 0.36 ± 0.44 0.11
d
A/EI
SOP remov al efficie ncy (%) 98.5 ± 1.3 95.7 ± 1.8 83.5 ± 2.2 88.6 ± 1.4 97.1 ± 1.6 78.9 ± 1.9 95.5 ± 1.2 98.2 ± 1.4 90.7 ± 1.3 74.5 ± 1.6
te
Regi me
DO Influ Efflue (m ent nt g/L type SOP ) (mg/L )
CO
N
18.1 ± 0.72 20.3 ± 1.31 19.5 ± 1.28 23.2 ± 1.67 21.7 ± 1.42 19.3 ± 0.84 28.7 ± 2.62 23.1 ± 2.27 31.4 ± 2.86 35.9 ± 3.41
us
P
Results are the averages and their standard deviations in triplicate tests.
24
Page 24 of 35
Table 2‐ Activities of PPX and PPK and bacterial populations in A/EI and A/O SBRsa
A/O
a
synthetic
1
synthetic
3
synthetic
5
synthetic
1
real
0.5
real
3
real
0.5
real
23.7 ± 3.6 19.8 ± 2.9
14.3 ± 3.2
ip t
5
27.2 ± 5.1
16.5 ± 4.4
16.8 ± 3.6
cr
synthetic
25.3 ± 4.4 21.2 ± 3.5
14.7 ± 2.8 17.1 ± 3.4
29.6 ± 4.7
12.4 ± 3.1
33.7 ± 5.5
10.7 ± 2.6
23.3 ± 3.4 19.8 ± 3.1
13.2 ± 2.9 18.5 ± 2.7
The data reported are the averages and their standard deviations in triplicate tests. Percentage to all bacteria (EUBmix probe). c The unit is μmol pnitrophenol/(min∙mg protein). d The unit is μmol NADPH/(min∙mg protein). b
Ac ce p
488 489 490 491 492
3
us
A/EI
synthetic
Bacterial populationsb PAOmix (%) GAOmix (%) 32.5 ± 5.4 12.1 ± 3.3
an
A/O
1
Enzyme activities PPXc PPKd 0.029 ± 0.278 ± 0.003 0.005 0.014 ± 0.169 ± 0.004 0.003 0.008 ± 0.113 ± 0.003 0.004 0.031 ± 0.216 ± 0.003 0.005 0.037 ± 0.283 ± 0.005 0.007 0.014 ± 0.121 ± 0.003 0.004 0.024 ± 0.253 ± 0.002 0.006 0.027 ± 0.262 ± 0.003 0.005 0.034 ± 0.271 ± 0.005 0.007 0.009 ± 0.112 ± 0.003 0.008
M
A/EI
DO Influent (mg/L) type
d
Regime
te
487
25
Page 25 of 35
110 Aerobic
Influent
Setting
100
Extended idle
ip t
A/EI regime A/O process
cr
90
80 Influent
Anaerobic
us
SOP removal efficiency (%)
decanting
Setting
Aerobic
idle
decanting
1
3 DO level (mg/L)
an
70
M
497
5
498
Ac ce p
te
d
Graphical Abstract. This paper showed that the aerobic/extended-idle (A/EI) regime drove superior biological phosphorus removal than the conventional anaerobic/oxic (A/O) process at low DO level.
27
Page 26 of 35
498
Highlights: Low DO level was demonstrated to improve BPR in A/EI regime;
500
DO concentration could affect PPX and PPK activities;
501
The biomass cultured at low DO level contained more PAOs and less
502
GAOs;
503
A/EI regime drove better BPR than A/O process at both high and low
504
DO levels;
505
A/EI regime had a better tolerance to oxygen-limited condition than
506
A/O process.
an
us
cr
ip t
499
Ac ce p
te
d
M
507
28
Page 27 of 35
Figure 1
7.8
6 Aerobic period
Settling/decanting periods
Idle period pH (1 mg/L) pH (3 mg/L) pH (5 mg/L) DO (1 mg/L) DO (3 mg/L) DO (5 mg/L)
DO (mg/L)
ip t cr
7.4
120
180
240 300 Time (min)
360
an
60
420
480
2
0
ce pt
ed
M
0
us
7.2
7.0
4
Ac
pH
7.6
Page 28 of 35
Figure 2
20 1 mg/L DO 3 mg/L DO 5 mg/L DO
cr
ip t
12
8
us
SOP (mg/L)
16
an
4
0
SOP uptake
Idle SOP release
Ac
ce pt
ed
M
Aerobic SOP release
Page 29 of 35
Ac c
ep te
d
M
an
us
cr
ip t
Figure 3
Page 30 of 35
Ac c
ep te
d
M
an
us
cr
ip t
Figure 4
Page 31 of 35
Figure 5
3
2
Aerobic period
360
420
0 480
Idle period 6 SOP Acetate PHB 5 PHV PH2MV PHAs 4 Glycogen
15
10
3 2
5 1
0
0
e5
20
2
180 240 300 Time (min)
Aerobic period
420
10
1
60
120
0 480
Idle period
180 240 300 Time (min)
Ac
0
360
420
SOP (mg/L)
0
d5
50
4
40
3
2
1
0
6 5
30
20
4
f5
50
3 2 1 0 480
3
2
120
180
240 300 Time (min)
360
420
0 480
Idle period
Aerobic period
6
5 SOP Acetate 4 PHB PHV PH2MV 3 PHAs Glycogen 2
10
0
4
60
Anaerobic period
0
ed
15
0
360
SOP Acetate PHB PHV PH2MV PHAs Glycogen
5
0
120
ce pt
3
60
SOP (mg/L)
Acetate (mmol-C/L)
4
0
0
1
M
1
10
0
60
120
Anaerobic period
180
1
240 300 Time (min)
360
420
0 480
Idle period
Aerobic period
6 5 SOP Acetate PHB 4 PHV PH2MV 3 PHAs Glycogen
40
30
20
2
1
10
0
0
1
0
60
120
180
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
180 240 300 Time (min)
SOP (mg/L)
Acetate (mmol-C/L)
4
120
1
240 300 Time (min)
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
20
60
2
360
420
0 480
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
c5
0
20
ip t
0
30
SOP (mg/L)
0
2
5 SOP Acetate 4 PHB PHV PH2MV 3 PHAs Glycogen
cr
1
3
6
us
1
40
Idle period
Aerobic period
an
5
4
Anaerobic period
SOP (mg/L)
2
50
Acetate (mmol-C/L)
3
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
10
b5
Acetate (mmol-C/L)
15
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
2
6 SOP Acetate PHB 5 PHV PH2MV PHAs 4 Glycogen
Acetate (mmol-C/L)
3
Idle period
PHB,PHV,PH2MV,PHAs,Glycogen (mmol-C/g-VSS)
Acetate (mmol-C/L)
4
Aerobic period 20
SOP (mg/L)
a5
Page 32 of 35
Figure 6
4
6
60
120
180 240 300 Time (min)
Aerobic period
360
420
Idle period
6
4
4
0
2.5
60
120
180
Aerobic period
240 300 Time (min)
2.0
1.5
360
420
480
Idle period 0.5 mg/L DO 1 mg/L DO
1.0
60
120
180 240 300 Time (min)
360
420
0 480
ed
0
0.5
0.0
0
60
120
180
240 300 Time (min)
360
420
480
ce pt
0
2
5
M
2
10
Ac
PHAs (mmol-C/g-VSS)
0.5 mg/L DO 1 mg/L DO
15
0
480
0.5 mg/L DO 1 mg/L DO
20
us
0
Glycogen (mmol-C/g-VSS) PHB PHV and PH2MV (mmol-C/g-VSS)
0
Idle period
ip t
8
Aerobic period
cr
0.5 mg/L DO 1 mg/L DO
12 SOP (mg/L)
25
Idle period
an
Aerobic period
PHB PHV and PH2MV (mmol-C/g-VSS)
16
Page 33 of 35
ip t cr
This study a
A/EI
1 3 5 1 3 5
Smolders et al. [26]
A/O
Glydeg/VFAup (mmol-C/mmol-C)
PHAssyn/VFAup (mmol-C/mmol-C)
PHB (%)
PHV (%)
PH2MV (%)
0.048±0.003
0.42±0.04
71.6±2.31
11.3±1.25
17.1±1.84
0.066±0.009
0.23±0.03
68.7±2.53
20.8±1.38
10.5±0.76
0.089±0.007
0.14±0.01
52.5±1.37
35.0±1.21
12.5±0.97
0.17±0.03
0.33±0.05
23.7±1.16
45.6±2.05
30.7±2.21
0.30±0.06
0.13±0.02
0.49±0.07
78.4±2.34
14.4±1.09
7.2±0.44
0.20±0.05
0.19±0.03
0.33±0.04
54.5±2.06
46.6±2.15
1.9±0.12
0.48
0.50
1.33
90
10
0
0.086±0.006 0.071±0.007 0.064±0.005 0.21±0.03
ep te
A/O
Prel/VFAup (mmol-P/mmol-C)
4
an
DO (mg/L)
M
Regime
d
Study
us
Table 3-Comparisons of stoichiometries and percentages of PHB, PHV and PH2MV in total PHAs between the aerobic period and conventional anaerobic period (acetate as carbon source)
Ac c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Pereira et al. [27]
A/O
2
0.16
0.69
1.47
71.4
28.6
0
Liu et al. [28]
A/O
6
0.45
0.78
1.47
75.8
20.0
4.2
A/O
6
0.57
0.53
1.30
88
12
0
A/O
-
0.96
0.48
1.24
89
9
2
Lu et al. [30]
A/O
2
0.62
0.46
1.26
94
6
0
Winkler et al. [25]
A2/O
6.25
0.15
0.71
1.11
77.3
22.7
0
Filipe et al. [18] Yagci et al. [29]
Page 34 of 35
ip t cr
d
M
an
us
The data reported are the averages and their standard deviations calculated from a long time operation.
ep te
a
Ac c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Page 35 of 35