Accumulation and isolation of simultaneous denitrifying polyphosphate-accumulating organisms in an improved sequencing batch reactor system at low temperature

International Biodeterioration & Biodegradation 100 (2015) 140e148

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Accumulation and isolation of simultaneous denitrifying polyphosphate-accumulating organisms in an improved sequencing batch reactor system at low temperature Shuli Liu a, b, Jianzheng Li a, b, * a b

School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 October 2014 Received in revised form 29 January 2015 Accepted 4 February 2015 Available online 12 March 2015

The paper studied accumulation of denitrifying polyphosphate-accumulating organisms (DNPAOs) by using an improved sequencing batch reactor (SBR) system in which anaerobic-aerobic-anoxic (A-O-A) and anaerobic-anoxic (AeA) are at low temperature (15
C). Results indicated that it was feasible to accumulate DNPAOs with nitrogen (N) and phosphorus (P) removals concurrently in an improved SBR system. Moreover, a DNPAOs strain was isolated from the accumulation and was identified as Acinetobacter sp. J6. Important factors that affect N and P removal efficiency of strain J6 were investigated, including temperature, pH and total phosphate (TP) concentration. Strain J6 had the highest N and P removal rates under original pH 8.0, TP concentration 4.16 mg L1 and 15
C. The purification culture of strain J6 contributed to the germplasm resources for a full-scale biological N and P removal system to denitrify and remove phosphate simultaneously. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Denitrifying polyphosphate-accumulating organisms Low temperature Nitrogen and phosphorus removal efficiency Accumulation Isolation Identification

Introduction Nitrogen (N) and phosphorus (P) are both key nutrients that stimulate the growth of algae and toxiccyano bacteria in natural water bodies, threatening drinking water safety and human health (Latif et al., 2011; Zhang et al., 2011; Bassin et al., 2012; Kim et al., 2013; Li et al., 2014). Since they are main pollution sources, removing N and P from wastewater has attracted extensive attention around the world (Hale et al., 2005; Chiu et al., 2007; Liu et al., 2010). According to conventional theory, N and P removals are function of different microorganisms in a wastewater treatment system (Liu et al., 1996; van Veldhuizen et al., 1999; Lee et al., 2003). Thus anaerobic denitrification unit and aerobic compartment of P removal are included in most wastewater treatment processes. Usually the residual sludge discharge is the only way for P removal. It suggests that the more P will be removed when the more P is

* Corresponding author. School of Municipal and Environmental Engineering, Harbin Institute of Technology, Huanghe Road 73, Nangang District, Harbin 150090, China. Tel./fax: þ86 45186283761. E-mail addresses: [email protected] (S. Liu), [email protected] (J. Li). http://dx.doi.org/10.1016/j.ibiod.2015.02.003 0964-8305/© 2015 Elsevier Ltd. All rights reserved.

absorbed by activated sludge. To enhance the capacity of P absorption, the aerobic sludge has to be recycled into the anaerobic unit for releasing more P, where denitrification also occurs simultaneously. Both denitrification process and P release of polyphosphate accumulating organisms (PAOs) require appropriate chemical oxygen demand (COD). Nitrate nitrogen (NO3  -N) or nitrite nitrogen (NO2  -N) had adverse effect on P release (Akin and Ugurlu, 2004; Oehmen et al., 2007; Wu et al., 2010; Park et al., 2011). So contradictions between denitrification and P release make it difficult to achieve N and P removals simultaneously in the same reaction fraction only by means of operation control and condition maintenance in treatment plants. In biological N and P removal system, microorganism groups that are mainly responsible for P removal are PAOs. Its metabolism process includes two steps. Under anaerobic conditions, PAOs would take up carbon sources such as preferably simple and short volatile fatty acids and store them as carbon polymers called polyb-hydroxybutyrates (PHBs or PHAs) (Jenkins and Tandoi, 1991; Merzouki et al., 2005). In the process, intracellular P was released by PAOs. Under aerobic or anoxic conditions, oxygen is used as only electron acceptors in respiratory chain (Mino et al., 1998; Blackall et al., 2002; Seviour et al., 2003; Zeng et al., 2003; McIlroy and

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 et al., 2011). At the same time, PAOs absorbed P Seviour, 2009; Taya from wastewater. NOx  -N can decrease P absorption of PAOs by impeding P release. Moreover, PAOs is one COD competitor of denitrifying bacteria that was responsible for removing NOx  -N. So in order to accumulate PAOs and reach up to the standards of effluent N and P, it is necessary to add carbon resource into the system. However the discovery of denitrifying polyphosphateaccumulating organisms (DNPAOs) in enhanced biological phosphorus removal (EBPR) process offered a proper solution to solve the problems associated with COD limitation (Seviour et al., 2003; Wang et al., 2009). DNPAOs have parrallel metabolic mechanism with PAOs except for their performance to use NO3  -N or NO2 -N instead of oxygen as electron acceptors. This physiological characteristic alleviate the NOx  -N suppression of P release and the competition for COD between P release and denitrification. And N and P removals simultaneously occurred in the same cell (KerrnJespersen and Henze, 1993; Seviour et al., 2003; Wang et al., 2009). So the studies on isolation, identification of DNPAOs and their characteristics of N, P removals have been focused in wastewater treatment fields due to their advantages over traditional PAOs (Seviour et al., 2003; Zeng et al., 2003; Wang et al., 2009). Hiraishi et al. (1998), Wagner et al. (1994) and Ahn et al. (2007) have reported that some DNPAOs such as Acinetobacter, Betaproteobacteria and Actinobacteria obtained high enrichment. G.W. FUHS has isolated DNPAOs successfully (Fuhs and Chen, 1975). Moreover, M. Sarioglu applied the pure culture of DNPAOs on biological P removal in a sequencing batch reactor (SBR) (Sarioglu, 2005). But most of studies were performed at room temperature (Fuhsand and Chen, 1975; Sarioglu, 2005), few studies on DNPAOs isolation at low temperature were reported. As an important index of assessing the overall efficiency of a biological treatment process, temperature not only influences metabolic activities of microbial population, but also it has a profound effect on gas-transfer rates and settling characteristics of biological solids. Every functional organism has its proper temperature range, and temperatures below its optimum range have more significant effects on growth rate than those above its optimum range, because activities of microorganisms will sharply decrease with decreasing temperature. The reports showed room temperature is suitable for N and P removal microorganisms (Mulkerrins et al., 2004; Li et al., 2010;  et al., 2012). So it is more difficult to accumulate and Gabarro isolate DNPAOs for simultaneous N and P removals. And there are limited DNPAOs resources at low temperature. Thus, this study aimed to accumulate DNPAOs in a SBR reactor operated by two stage performance (anaerobic-aerobic-anoxic (AO-A) and anaerobic-anoxic (AeA)) at 15
C. To isolate and identify

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efficient DNPAOs strain with high N and P removals simultaneously from accumulated DNPAOs. To determine optimal conditions of isolated strain with favourable N and P removal efficiency. Important factors (temperature, pH, total phosphate (TP) concentration and rotating speed) affecting N and P removal efficiency of isolated strain were investigated in the present paper. Materials and methods Inoculum and domestic wastewater The seed sludge applied for DNPAOs accumulation was taken from the secondary clarifier sludge in a local sewage treatment plant in Harbin, China. Total suspended solids (TSS), volatile suspended solids (VSS), sludge volume index (SVI) and pH of the sludge were 7.27 g L1, 5.17 g L1, 93.48 and 7.3, respectively. Synthetic wastewater was prepared for DNPAOs accumulation in different nutrients loadings (COD: 200, 350 mg L1, COD: ammonium (NH4 þ -N): TP ¼ 200: 15: 2.5). The composition of wastewater (COD 200 mg L1) was as follow in g L1: 0.3 CH3COONa, 0.09 NH4Cl, 0.018 KH2PO4, 0.01 EDTA. The trace element contained following ingredients (g L1): 3 FeSO4$7H2O, 0.026 MnSO4$H2O, 0.05 CoCl2$6H2O, 0.007 CuSO4$H2O, 0.05 ZnSO4$7H2O, 0.02 H3BO3. 2 mL trace element was added into 1 L wastewater. The main water quality indexes of domestic wastewater were as follows. The concentration of COD, NH4 þ -N, NO3  -N, NO2  -N and TP was 180 ~ 350, 23 ~ 65, 0.2 ~ 1.5, 0 ~ 0.2 and 2.8 ~ 6.4 mg L1, respectively. Reactor design and operating conditions A lab-scale SBR had a total reactor volume of 10 L and a reaction volume of 6 L that was shown in Fig. 1. The operation condition were as follows: mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS) and temperature were 3.357 g L1, 2.335 g L1 and 15
C, respectively. Excess sludge was regularly removed to keep sludge retention time within the 15th day. The operation on DNPAOs enrichment included two processes in an improved SBR system: 1. A-O-A SBR, PAOs accumulation; 2. AeA SBR, DNPAOs were enriched from PAOs accumulation. The detailed information was formulated in Table 1. In A-O-A SBR, experimental start-up carried out stage 1 and original A-O-A SBR (stage 2). This process (stage 2) was improved by adding an anoxic phase to eliminate adverse effect of residual NOx  -N on P release in a new cycle, which was superior to traditional A-O SBR (stage 1). In the enhanced A-O-A SBR (stage 3), HRT was shorted and influent COD loading was increased by 1.75 times

Fig. 1. Schematic drawing of the SBR system.

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Table 1 Operating model, stage and condition of each cycle in the SBR. Operating model

Anaerobic-aerobicanoxic

Anaerobic-anoxic a b

Stages

Stage Stage Stage Stage Stage Stage

1a 2a 3a 4b 5a 6a

Cycles

1 ~ 13 15 ~ 31 33 ~ 51 53 ~ 59 1 ~ 15 17 ~ 33

Cycle time (h)

In a cycle Influent COD (mg/L)

Feeding time (min)

Anaerobic phase (h)

Aerobic phase (h)

Anoxic phase (h)

Settling phase (min)

Discharging time (min)

Post-aeration phase (min)

12 12 10 10 8 8

200 200 350 350 350 350

10 10 10 10 10 10

3 3 2 2 2 2

8 7 6 6 e e

e 1 1 1 5 5

40 40 30 30 30 30

10 10 10 10 10 10

e e 10 10 10 10

Fed with synthetic wastewater. Fed with domestic wastewater.

to increase N and P removal efficiency of the system. In order to investigate the feasibility and stability of SBR system with respect to N and P removals for domestic sewage, the process (stage 4) for treating domestic sewage was carried out. In A- A SBR, to accumulate DNPAOs from PAOs accumulation, nitrate instead of oxygen was added into the system as electron acceptors at the beginning of the anoxic phase. And the average nitrate concentration was 6.57 mg L1 in stage 5. In stage 6, the average nitrate concentration was increased to 9.06 mg L1 to enhance N and P removal efficiency of DNPAOs accumulation. In the operations above, DO was maintained less than 0.5 mg L1, 2.0 ~ 2.5 and 0.5 ~ 1.0 mg L1 in the anaerobic, aerobic and anoxic phases, respectively.

Microbial isolation and culture mediums According to traditional bacteria separation methods, sludge sample from AeA SBR system was divided into two average portions. One sample was incubated in nutrient medium for 15 d while the other was not disposed at all. The preparation of original bacterium suspension consisted of mixed 1 mL sample above and 9 mL sterile water. Subsequently two suspensions were diluted into various gradients of different concentrations such as 102, 103, 104, 105 in accordance with doubling dilution method. Each sample of one concentration was conducted in triplicates for DNPAOs isolation and screening by means of different special culture mediums. The mediums included enrichment medium, isolation mediums with different carbon resource, rich phosphorus medium and phosphorus deficiency medium (Merzouki et al., 1999). And the formula of mediums were detailed in supplementary Table S1. Isolated strains that had similar morphological characteristics were cultured alternately in the special solid and liquid medium for 3 ~ 4 times. Purified strains were used for P removal test after 48 h, and high-efficiency strains (P removal rates were no less than 50%) were selected. In N removal test, N removal rates of the strains in the medium containing NO3  -N were detected after 48 h. Besides, morphological characteristic particles including PHBs and metachromatic granules of optimal DNPAOs strain were detected in. In order to ensure the accuracy of data, all samples were conducted in triplicates and the reported results were the average values. The error was not more than 5%.

Microbial community analysis and identification of DNPAOs strain DNA isolation DNA isolation was operated by the standard of PowerSoil® DNA Isolation Kit (MoBio Laboratories Inc, USA). A band for corresponding DNA was observed on 1.5% agarose gel under UV at 254 nm.

PCR-DGGE process and sequences analysis The V3 region of 16S rDNA were amplified by PCR using universal bacterial primers (BSF8, 50 -AGAGTTTGATCCTGGCTCAG-30 and BSR534, 50 -ATTACCGCGGCTGCTGG-30 with a GC clamp, BSF341, 50 -CCTACGGGAGGCAGCAG-30 , BSF907, 50 - CCGTCAATTCMTTTGAGTTT-30 ). The PCR amplification was conducted in a 50 mL system containing 5 mL 10  Ex Taq buffer, 4 mL dNTP mixture (2.50 mM), 1 mL forward primer (20 mM), 1 mL reverse primer (20 mM), 2.5 ng DNA template, and 0.15 U Ex Taq DNA polymerase (Takara, Dalian, China). The PCR conditions were as follows: initial denaturation (5 min at 94
C), followed by 32 cycles of denaturation (1 min at 94
C), primer annealing (45 s at 45
C) and primer extension (1.5 min at 72
C), and final extension (7 min at 72
C). Amplified gene products were visualized on 1.5% agarose gel under UV at 254 nm. For DGGE analysis, PCR products (15 mL) were separated on polyacrylamide gels (8%) with a 40% ~ 60% linear gradient of denaturant (100% denaturant: 7 mol L1 urea and 40% formamide). The gel was conducted at 60
C in 1  TAE buffer at 100 V for 12 h by a Dcode Universal Mutation Detection System (Bio-Rad). Then the gel was stained using silver-staining method and visualized by the scanner (UMAX PowerLook 1000). Specific gel bands were selected and dissolved in 30 mL 1  TE buffer at 4
C overnight. 3 mL solution (DNA template) was reamplified. The PCR products were used for connecting PMD®18-T Vector and transformation in a medium containing Amp resistance, IPTG and X-gal as induction. The aimed strains were used for sequencing by Sangon Biotech. The gene sequences were analyzed against the NCBI database using BLAST N program packages and matched with known 16S rDNA gene sequences (http://blast.ncbi. nlm.nih.gov/Blast.cgi). Then a phylogenetic tree of the objective DNPAOs strain was constructed with MEGA5.0.

Analytical methods Main indexes such as NH4 þ -N, NO2  -N, NO3  -N, COD, TP and dry cell weight were analyzed according to standard methods (Clesscerl et al., 1998). TN was measured with (SHIMADZUVCPNTOC analysis meter, Japan). pH was measured with pH meter (DELTA 320, Switzerland). DO was measured with Dissolved Oxygen Meter for a certain frequency (Oxi730, Beijing). Microorganisms characteristics of sludge flora in different periods were observed Scanning Electron Microscope, SEM (KYKYe2800B, Beijing). Surface characteristics of isolated DNPAOs strain and its intracellular unique inclusion were detected with Atomic Force Microscope, AFM (Bioscope™, USA) and Transmission Electron Microscope, TEM (TEM1400, Japan), respectively. The bacteriaegrowth curve of DNPAOs strain was measured by OD600 value with ultraviolet spectrophotometer (UV2300, Shanghai) and partial physiological and biochemical reactions of the DNPAOs

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strain were operated by bacterial micro reagents of biochemical reaction. Results and discussion DNPAOs accumulation and its efficiency for N and P removal in a SBR A-O-A SBR start-up and its efficiency analysis at low temperature In order to prove PAOs accumulation in an improved A-O-A SBR, NH4 þ -N, NOx  -N, COD and TP concentrations were measured and the results were shown in Fig. 2. To eliminate the adverse effect of residual NOx  -N on P release in next cycle, the improved system was operated as described in original A-O-A SBR with adding an anoxic phase after the aerobic phase (Fig. 2-stage 2) and Table 2. It indicated the reverse effect of the residual NOx  -N on P release in next cycle was eliminated. The average concentrations of NO2  -N and NO3  -N gradually increased, stabilized at 4.13, and 9.08 mg L1 at the end of the aerobic phase in sequential cycles. And the corresponding values were both low at 0.69 and

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1.65 mg L1 at the end of the anoxic phase, which kept low NOx  -N concentration in next cycle. Based on obviously improving NH4 þ -N removal, NO2  -N and NO3  -N producing rates in the aerobic phase increased to 5.95 and 12.80 mg g1-MLVSS d1 in stage 2, while the corresponding values were only 3.34 and 6.21 mg g1-MLVSS d1 in stage 1. Moreover NO2 -N and NO3  -N removal rates in the anoxic phase were as high as 34.56 and 75.01 mg g1-MLVSS d1. So the added anoxic phase eliminated NOx  -N accumulation, which responded to the effective reports in the studies (Wu et al., 2010;  et al., 2011). It indicated A-O-A SBR operation could improve Taya NOx  -N removal. In addition, the average concentration of effluent TP decreased to 0.61 mg L1 (stage 2) from 1.85 mg L1 (stage 1), and the P release rate in the anaerobic phase increased to 40.51 mg g1MLVSS d1 (stage 2) from 15.46 mg g1-MLVSS d1 (stage 1), the total P uptake rate in the aerobic and anoxic phases increased to 19.61 mg g1-MLVSS d1 (stage 2) from 5.08 mg g1-MLVSS d1 (stage 1). The relation between the P uptake rate and the P release rate was modeled (Fig. 3a). The results showed that the P release rate was in a linear relationship with the P uptake rate no matter in

Fig. 2. Induces analysis including NH4 þ -N, NO2  -N, NO3  -N, TP and COD concentrations in anaerobic-aerobic-anoxic (A-O-A) SBR Annotation: operating models of stage 1, stage 2, stage 3 and stage 4 are described in A-O-A SBR of Table 1.

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Table 2 Nutrients removal efficiency in the SBR under different operating models. Operating model

Stages

Anaerobic-aerobic- Stage 1 anoxic Stage 2 Stage 3 Stage 4 Anaerobic-anoxic Stage 5 Stage 6

Cycles

NH4 þ -N NO2  -N (mg g1-MLVSS d1) NO3  -N (mg g1-MLVSS d1) TP (mg g1-MLVSS d1) COD removal removal Aerobic Anoxic Aerobic Anoxic Anaerobic release rate Aerobic and anoxic producing rate removal rate producing rate removal rate removal rate

9 ~ 13 27 ~ 31 47 ~ 51 55 ~ 59 1 ~ 15 17 ~ 33

85.12% 97.84% 98.26% 98.91% e e

e 34.56 39.33 36.49 e e

3.34 5.95 7.68 7.29 e e

traditional A-O SBR (stage 1) or original A-O-A SBR (stage 2), so more P released from the sludge, more P would be absorbed by the sludge. It would contribute to high P removal. It is agreed the P uptake in the aerobic zone is directly related to the quantity of P release in the anaerobic zone at temperatures between 15
C and 20
C described in some models of biological phosphorus removal (Helmer and Kunst, 1998). In the enhanced A-O-A SBR (Fig. 2-stage 3), the concentration of effluent NH4 þ -N was less than 0.46 mg L1, the NOx  -N concentration gradually increased in the aerobic phase and the NOx  -N concentration was low than 0.68 mg L1 in the anoxic phase at the stable operation even the loadings of influent nutrients were

a

20 18 16 14

-1

-1

The P uptake rate/ (mg·g -MLVSS·d )

22

12 10 8

y=0.4724 x + 0.1668 2 R =0.9973

6 4 2 0

0

4

8

12

16

20

24

28

32

36

-1

40

44

48

-1

The P release rate/ (mg·g -MLVSSd )

17.5

b

17.0 16.5

-

-1

-1

Utilized NOX -N/ (mg·g -MLVSS·d )

18.0

16.0 15.5 15.0

y=1.087 x - 6.49 2 R = 0.8255

14.5 14.0 13.5 13.0 25.0

25.5

26.0

26.5

27.0

27.5 -1

28.0

28.5

29.0

-1

Consumed P/ (mg·g -MLVSS·d ) Fig. 3. a: Relationship between the P uptake rate and the P release rate in anaerobicaerobic-anoxic (A-O-A) SBR. b: Relationship between utilized NOx  -N and consumed P in anaerobic-anoxic (AeA) SBR.

6.21 12.80 17.26 20.38 e e

e 75.01 98.04 114.24 11.98 17.53

12.87 41.23 51.21 52.87 56.29 51.00

5.08 19.61 22.95 23.19 73.16 69.01

68.09% 93.51% 93.38% 93.35% 87.27% 93.32%

increased. The TP removal increased and stabilized at 84.28% with increasing TP concentration. The P release rate in the anaerobic phase increased to 51.21 mg g1-MLVSS d1 and the total P uptake rate in the aerobic and anoxic phases was the highest at 22.95 mg g1-MLVSS d1. And the COD removal was more than 90%. Hence it indicated A-O-A SBR operation had achieved effective nutrients removal and PAOs were well accumulated. As it was shown in Fig. 2-stage 4, PAOs had efficient TP, TN, COD removals, and the corresponding values were 83.59%, 89.79%, 93.31% for treating domestic sewage, which indicated PAOs accumulation had stable removal efficiency of nutrients. The operation of AeA SBR and its efficiency analysis In order to enrich DNPAOs in the PAOs, the system was converted into AeA SBR operation, especially adding nitrate into the system at the beginning of anoxic phase. The relation between utilized NOx  -N and consumed P was modeled in Fig. 3b. It indicated utilized NOx  -N and consumed P had well linear relationship. As Fig. 4-stage 5 showed, the average concentration of adding NO3  -N for DNPAOs accumulation was at 6.57 mg L1, the average concentrations of effluent NO2  -N, NO3  -N, TP and COD were 0.47, 0.84, 0.77 and 46.18 mg L1 at the end of the anoxic phase. The average NO3  -N removal was 87.41%. The P release rate in the anaerobic phase was 56.29 mg g1-MLVSS d1 and the P uptake rate in the aerobic and anoxic phases was 73.16 mg g1-MLVSS d1. In the AeA SBR (Fig. 4-stage 6), when the average concentration of adding NO3  -N was increased to 9.06 mg L1, the utilized NOx  -N rate increased to 17.53 mg g1-MLVSS d1 from 11.98 mg g1-MLVSS d1 with increasing NO3  -N. So increasing NOx  -N and TP removals indicated that the proportion of DNPAOs in the system increased after AeA SBR stages. Moreover, the utilized NOx  -N rate had a positive linear correlation with the consumed P in the anoxic phase (Fig. 3b). This was because that main P removal was removed by DNPAOs. At the same time, adding NO3  -N was consumed as electron acceptor, resulting to proportional NOx  -N and P removals. It meant that more DNPAOs were accumulated. The system had favorable N and P removals at the stable operation, where the average concentrations of effluent NO2  -N, NO3  -N, TP and COD were 0.47, 0.50, 0.71 and 24.05 mg L1. DNPAOs were accumulated that had efficient denitrifying and dephosphorization utilizing operations including A-O-A SBR and AeA SBR. And accumulated DNPAOs could be potential sources for the isolation of efficient strains. The microbial community analysis in the system In order to confirm DNPAOs accumulation further, microbial community dynamics was analyzed by PCR-DGGE and SEM. The results were shown in Fig. 5, Table 3 and supplementary Fig. S2. In Fig. 5, the marks 0, 1, 2, 3 and 4 represents stages 1, 2, 3, 4 in Fig. 2 and stage 6 in Fig. 4. Nocardiopsis sp. (band a) was main

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Fig. 4. Induces analysis including NO2  -N, NO3  -N, TP and COD concentrations in anaerobic-anoxic (AeA) SBR Annotation: operating models of stage 5, stage 6 are described in AeA SBR of Table 1.

dominant bacteria and occupied major amount, which played a significant role in efficient N removal in all stages. However, the amount of Bacterium sp. (band f) gradually decreased with operating stages, and there is hardly detailed information about its N or P removal. After the enhanced A-O-A SBR (Fig. 2 stage 3), the proportions of Sphingomonas sp. (band b), Paenibacillus sp. (band d) and Actinobacterium sp. (band e) all increased, some of which were identified to belong to DNPAOs. In stage 6 of AeA SBR, besides the dominant bacteria Sphingomonas sp. (band b) and Paenibacillus sp. (band d), Acinetobacter sp. (band c) played an important role in the accumulated microorganisms, which was reported to be one typical DNPAOs. Acinetobacter was widely reported to be responsible for P uptake (Kavanaugh, 1991). In the study on biological P removal in a SBR by using pure cultures, Acinetobacter was the predominant species and high P removal efficiency was obtained (Sarioglu, 2005). This showed DNPAOs could be abundantly accumulated in the system after AeA SBR. As the labels in Fig. S2 was the same to those in Fig. 5, the SEM of accumulated microorganisms at different stages in the system indicated that the amount of sphaerita and brevibacterium gradually increased with increasing N and P removals. After A-O-A SBR, the amount of small sphaerita obviously rose and the lamellar

Table 3 Taxonomic identification of the sequences from DGGE bands.

Fig. 5. DGGE profiles of bacteria of different stages in SBR system Annotation: operating models of 0 (stage 1), 1 (stage 2), 2 (stage 3), 3 (stage 4) in A-O-A SBR and 4 (stage 6) in AeA SBR are described in Table 1.

Band

Closest relative

Identity (%)

Access no.

a b c d e f

Nocardiopsis sp. AM8 Sphingomonas sp. PY84T Acinetobacter sp. Dui-5 Paenibacillus barengoltzii THWCS11 Bacterium CB1 Bacterium Lim C-1-J-55B

100% 100% 100% 100% 97% 97%

AM236241 FJ799016 EF031061 GQ284358 JN983821 JF900790

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decreased in the system. In AeA SBR, the concavo-concave round bacteria and brevibacterium occupied major amount, and they existed in the form of cluster. The accumulated microorganisms above were used for DNPAOs isolation. The identification of an isolated strain From the accumulated sludge, 6 DNPAOs strains were isolated and gained by the methods described in “Microbial isolation and culture mediums”. The strain with high N and P removal efficiency was selected for the further study and it was named strain J6. In order to identify strain J6, Biological characteristics and 16 S rDNA sequencing of strain J6 were analyzed. Biological characteristics of strain J6 Morphological characteristics of strain J6. From AFM and TEM photos (Fig. S 3a, Fig. S 3b), strain J6 is a typical sphaerita, and it has specific intracellular granule including PHB or poly-phosphate as DNPAOs. These obvious granules exist around cell nucleus. Physiological and biochemical characteristics of strain J6. Main physiological and biochemical reactions of strain J6 were described in the supplementary Table S4 and the growth curve of strain J6 was drawn (Fig. S5). In the beginning of growth cycle, TP concentration firstly experienced descent and subsequently increased to the original level with increasing OD600, decreasing TP was used for cells synthesis and subsequent P release of cells. In next logarithmic phase and stationary phase, TP concentration in medium decreased continuously due to cells absorption after P release. The TN gradually decreased in the whole growth cycle. Phylogenetic analysis of strain J6 The production for 16S rDNA PCR amplification of strain J6 was applied for sequencing, and nucleotide homology comparison between target sequencing column and 16S rDNA sequences in the GenBank was made through the BLAST procedures. Phylogenetic tree of strain J6 was constructed (Fig. 6). The result showed strain J6 belonged to the Acinetobacter sp. with maximum homology. Effects analysis on denitrifying phosphorus removal of strain J6 In order to enhance N and P removal efficiency of strain J6 further, major factors including temperature, pH, TP concentration

and rotating speed affecting TN and TP removals of strain J6 were optimized. Effects of temperature on growth and denitrifying phosphorus removal of strain J6 Temperature influenced metabolic activities of microbial population, especially temperatures below the optimum had more significant effects on growth rate than those when temperatures were above the optimum. Many studies take references of different suitable temperature of different function microorganism at respective condition (Mulkerrins et al., 2004; Li et al., 2010;  et al., 2012). Thus, effects of temperature on TN and TP Gabarro removal rates of strain J6 were tested in this study. The results (Fig. 7a) showed that strain J6 had a stable TN and TP removal efficiency, it had the highest TN and TP removal rates and the corresponding values were 2.75 and 13.11 mg g1-dry cell d1 at 15
C. The dry cell weight was 1.11 g L1. Agreed with the conclusion mentioned above, TN and TP removals both sharply decreased with decreasing temperature, and strain J6 had no growth at much lower temperature (8
C or 10
C). When temperature was increased to 20
C or 25
C, TP removal hardly decreased and TN removal slightly decreased, strain J6 had no obvious growth with dry cell weight at 1.08 and 1.03 mg g1dry cell d1, respectively. So strain J6 had stable and favourable N and P removal efficiency at 15
C, which can be used for providing germplasm resources for the study on N and P removal at low temperature. Effects of pH on growth and denitrifying phosphorus removal of strain J6 As a critical factor in the N and P removal system, pH plays a significant role in the processes such as P release and VFAs uptake in the anaerobic phase, nitrification in the aerobic phase and P uptake or denitrification in the anoxic phase (Lee et al., 2001; Oehmen et al., 2007). For simultaneous N and P removals of DNPAOs, pH had irreplaceable effect on reaction efficiency. The results about effects of pH on TN and TP removal efficiency of strain J6 (Fig. 7b) showed that strain J6 had a relatively extensive range of pH for TN and TP removal rates. When pH ranged between 7.0 and 8.5, TN and TP removal rates were relatively high, especially TP removal rate reached the peak (1.64 mg g1-dry cell d1) as well as maximum TN removal rate was 16.21 mg g1-dry cell d1 when pH was 8.0. Moreover, the dry cell weight of strain J6 was

Fig. 6. Phylogenetic tree of strain J6 among the homologous genera.

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Fig. 7. Effects analysis on denitrifying phosphorus removal of strain J6. a: Temperature. b: pH. c: TP concentration d: Rotating speed.

0.82 g L1 at this condition. TP and TN removals in the systems were 83.69% and 69.25%. So optimal pH of strain J6 in the study is 8.0 for both maximum TN and TP removals. Effects of TP concentration on growth and denitrifying phosphorus removal of strain J6 To discuss effects of TP concentration on TN and TP removal efficiency, TN, TP removals and strain J6 growth were tested. The results (Fig. 7c) showed that TP and TN removals of the systems reached the peaks at 97.79% and 78.52%, and the dry cell weight of strain J6 was 1.11 g L1 when TP concentration was 4.16 g L1. The corresponding TP and TN removal rates were 4.22 and 13.11 mg g1-dry cell d1. The TP and TN removals decreased to 82.77% and 75.36%, the dry cell weight decreased to 0.87 g L1. The TP and TN removal rates were 1.26 and 12.59 mg g1-dry cell d1 when TP concentration was 2.16 mg L1. When TP concentration was increased to 9.46 mg L1, TP and TN removals decreased to 55.88% and 30.35%, and the dry cell weight was 0.73 g L1. The corresponding TP and TN removal rates were 3.73 and 5.07 mg g1dry cell d1. In DNPAOs cells, TN and TP removals simultaneously occurred by P uptake with either NOx -N or oxygen as electron acceptor. The properly increasing TP could enhance N and P removal efficiency at a certain extent. But too high TP decreased N, P removal efficiency and strain growth. The optimal TP concentration was 4.16 mg L1 for strain J6 in this study. Effects of rotating speed on growth and denitrifying phosphorus removal of strain J6 Rotating speed not only includes gas-transfer rate between aeration and liquid culture medium, but also it affects substancetransfer rate between cells and nutrients (Mulkerrins et al., 2004). The optimum of rotating speed (Fig. 7d) represented that TP removal rate, TN removal rate and strain growth had no obvious

difference from 120 to 140 rpm, corresponding values were 2.76 mg g1-dry cell d1, 13.20 mg g1-dry cell d1 and 1.11 g L1 at 120 rpm. The values were 2.77 mg g1-dry cell d1, 13.18 mg g1dry cell d1 and 1.11 g L1 at 140 rpm. High rotating speed (180 rpm) had little mass-transfer efficiency in the reaction system, resulting to low TN removal rate (6.35 mg g1-dry cell d1) and TP removal rate (1.29 mg g1-dry cell d1). Because there was no sufficient reaction time between cells and nutrients or the disruption of partial cells, resulting into decreasing TN and TP removal rates at high rotating speed. So strain J6 had optimum rotating speed from 120 to 140 rpm in this study. Conclusions Efficient N and P removal system was established successfully by the operation of improved A-O-A SBR and AeA SBR even at 15
C, which was deemed as lower critical threshold of temperature for denitrifying and dephosphatation. The system had stable N and P removals for treating domestic sewage. DNPAOs were successfully accumulated in the system and one DNPAOs strain (J6) of high N and P removal efficiency was isolated and identified. Strain J6 had the potential of efficient, simultaneous N and P removals. The highest TN and TP removal rates were achieved at this condition (pH 8.0, TP concentration 4.16 mg L1, rotating speed (120 ~ 140 rpm) and 15
C). Strain J6 was identified to belong to Acinetobacter sp. by phylogenetic analysis, which was proved to be responsible for P removal in activated sludge. Deeper metabolism exploitation of strain J6 at low temperature will be needed in further research. The successful operation of improved A-O-A SBR and AeA SBR at 15
C will be applied for N and P removal system in wastewater treatment fields. The accumulated microorganisms could be as popular inoculum in other reactors at low temperature. The isolated strain would provide certain germalasm resources for

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