Functional bacteria and process metabolism of the Denitrifying Sulfur conversion-associated Enhanced Biological Phosphorus Removal (DS-EBPR) system: An investigation by operating the system from deterioration to restoration

Water Research 95 (2016) 289e299

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Functional bacteria and process metabolism of the Denitrifying Sulfur conversion-associated Enhanced Biological Phosphorus Removal (DS-EBPR) system: An investigation by operating the system from deterioration to restoration Gang Guo a, b, c, Di Wu a, b, c, **, Tianwei Hao a, b, c, Hamish Robert Mackey d, Li Wei a, b, c, Haiguang Wang a, b, c, Guanghao Chen a, b, c, e, * a

Department of Civil & Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong, China Water Technology Laboratory, The Hong Kong University of Science and Technology, Hong Kong, China c Hong Kong Branch of Chinese National Engineering Research Center for Control & Treatment of Heavy Metal Pollution, The Hong Kong University of Science and Technology, Hong Kong, China d College of Science and Engineering, Hamad bin Khalifa University, Doha, Qatar e Beijing University of Civil Engineering and Architecture, Beijing, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 November 2015 Received in revised form 1 March 2016 Accepted 5 March 2016 Available online 8 March 2016

A sulfur conversion-associated Enhanced Biological Phosphorus (P) Removal (EBPR) system is being developed to cater for the increasing needs to treat saline/brackish wastewater resulting from seawater intrusion into groundwater and sewers and frequent use of sulfate coagulants during drinking water treatment, as well as to meet the demand for eutrophication control in warm climate regions. However, the major functional bacteria and metabolism in this emerging biological nutrient removal system are still poorly understood. This study was thus designed to explore the functional microbes and metabolism in this new EBPR system by manipulating the deterioration, failure and restoration of a lab-scale system. This was achieved by changing the mixed liquor suspended solids (MLSS) concentration to monitor and evaluate the relationships among sulfur conversion (including sulfate reduction and sulfate production), P removal, variation in microbial community structures, and stoichiometric parameters. The results show that the stable Denitrifying Sulfur conversion-associated EBPR (DS-EBPR) system was enriched by sulfate-reducing bacteria (SRB) and sulfide-oxidizing bacteria (SOB). These bacteria synergistically participated in this new EBPR process, thereby inducing an appropriate level of sulfur conversion crucial for achieving a stable DS-EBPR performance, i.e. maintaining sulfur conversion intensity at 15e40 mg S/L, corresponding to an optimal sludge concentration of 6.5 g/L. This range of sulfur conversion favors microbial community competition and various energy flows from internal polymers (i.e. polysulfide or elemental sulfur (poly-S2-/S0) and poly-b-hydroxyalkanoates (PHA)) for P removal. If this range was exceeded, the system might deteriorate or even fail due to enrichment of glycogen-accumulating organisms (GAOs). Four methods of restoring the failed system were investigated: increasing the sludge concentration, lowering the salinity or doubling the COD loading, non of which restored SRB and SOB activities for DS-EBPR; only the final novel approach of adding 25 ± 5 mg S/L of external sulfide into the reactor at the beginning of the anoxic phase could efficiently restore the DS-EBPR system from failure. The present study represents a step towards understanding the DS-EBPR metabolism and provides an effective remedial measure for recovering a deteriorating or failed DS-EBPR system. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Sulfur conversion Enhanced biological phosphorous removal Microbes and metabolism System deterioration and restoration

1. Introduction * Corresponding author. Department of Civil & Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong, China. ** Corresponding author. Department of Civil & Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong, China. E-mail addresses: [email protected] (D. Wu), [email protected] (G. Chen). http://dx.doi.org/10.1016/j.watres.2016.03.013 0043-1354/© 2016 Elsevier Ltd. All rights reserved.

The Enhanced Biological Phosphorus Removal (EBPR) system was developed for biological phosphorus removal from wastewater. In this system, polyphosphate-accumulating organisms

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(PAOs) (Kuba et al., 1993; Smolders et al., 1994) compete with glycogen-accumulating organisms (GAOs) for limited organic substrates under alternating anaerobic and aerobic or anoxic conditions (Oehmen et al., 2007; Bassin et al., 2012). Several studies have confirmed that PAOs are sensitive to high sulfate reduction (Yamamoto et al., 1991; Baetens, 2001), especially in the presence of high sulfate concentrations (Wu et al., 2013). The frequent application of sulfate coagulants in drinking water treatment works (Rauch and Kleidorfer, 2014), brackish water supply systems (Beer, 1999) and/or sulfate-laden groundwater supply systems often results in high sulfate concentrations in municipal wastewater (Lens et al., 1998; van Loosdrecht et al., 2012), rendering current EBPR technologies ineffective (Howarth and Paerl, 2008). In addition to high salt and sulfate limitations, warm water temperatures (>20
C) also enable GAOs to outcompete PAOs (Lopez-Vazquez et al., 2009). These situations are particularly commonplace in subtropical Hong Kong where seawater toilet flushing has been practiced on a large scale since 1958 (Tang et al., 2007; Leung et al., 2012). Inevitably this dual water supply system results in saline sewage featuring a high ratio of sulfate to chemical oxygen demand (COD; > 1.25 mg SO2 4 /mg COD). Leveraging these unique sewage features, the Sulfate reduction, Autotrophic denitrification and Nitrification Integrated (SANI®) system was successfully developed for the treatment of Hong Kong's saline sewage (Wang et al., 2009). Since 2012, this system has been further extended to biological phosphorus removal, i.e. a Denitrifying Sulfur cycle-associated EBPR (DSEBPR) process (Wu et al., 2014). Related sulfur studies have been reported very recently (Ginestet et al., 2015; Daigger et al., 2015). In this novel DS-EBPR system, poly-b-hydroxyalkanoates (PHA), polysulfide and elemental sulfur (poly-S2-/S0) are believed to form in sludge linked with carbon sources uptake and glycogen degradation under anaerobic conditions (P release phase), while under the subsequent anoxic conditions (P uptake phase), PHA and polyS2-/S0 are likely utilized for P uptake with glycogen replenishment and poly-S2-/S0 oxidized back to sulfate (Wu et al., 2014). However, little is known about the functional bacteria and metabolism of this new system except that conventional PAOs are not involved but enrichment of fermentative bacteria (e.g. Lactococcus-like species) and sulfate-reducing bacteria (SRB) species (e.g. Desulfobulus-like species and Desulfomicrobium-like species) are observed (Wu et al., 2014). Specifically, it is unknown whether and how these detected bacteria perform P release and P uptake, or if other bacteria are involved in the observed P removal. This study was specifically aimed at identifying the major functional microbes and studying the process metabolism of DSEBPR. Since bacterial genera in biological phosphate removal systems cannot be easily isolated in pure cultures for such an investigation (Seviour et al., 2003), we manipulated the system from stable operation to deterioration or even failure to investigate the functional microbes and possible system metabolism. We proceeded to monitor the shift in the microbial structures, determine changes in relevant anaerobic stoichiometric parameters and explore any relationship that may exist between sulfur conversion levels and P removal. In an EBPR system, many factors may cause EBPR deterioration and failure, such as types of influent carbon source, ratios of influent phosphorus to carbon, pH, and temperature (Oehmen et al., 2007; Lopez-Vazquez et al., 2009). Among these factors, sludge concentration was reported to be the key one (Henze et al., 2008; Morgenroth and Wilderer, 1999; Okunuki et al., 2004; Vaiopoulou et al., 2007). Therefore, various sludge concentrations were tested to drive a stable DS-EBPR system towards deterioration or failure. Then four operational factorsdsludge concentration, salinity, COD concentration, and sulfide leveldwere tested to

restore the failed system. 2. Material and methods 2.1. Reactor and operating conditions A lab-scale sequencing batch reactor (SBR) made from PVC with a maximum volume of 30 L was used in this study, as shown in Fig. S1 (Supplementary Information (SI) 1). The reactor was tightly sealed and continuously operated under dark conditions for 250 days, following the study of Wu et al. (2013). The reactor temperature was controlled at 30 ± 1
C by a water-bath heater to mimic the typical sewage temperature in Hong Kong. The reactor pH was kept between 7.2 and 7.8 by adding 0.5 N HCl and 0.5 N NaOH solutions alternatively when necessary. Inoculum of the reactor was taken from an anaerobic sludge digester at a local saline sewage treatment works. Following our previous study (Wu et al., 2013), this SBR was operated under alternating anaerobic and anoxic conditions at effective working volumes of 30 L or 20 L respectively, corresponding to the following cyclic operation conditions. Each operating cycle consisted of: i) feeding 15 L of synthetic sewage from Day 1 to Day 157 and 10 L of synthetic sewage from Day 158 to Day 250 for 10 min each cycle (see the next section for explanation), ii) an anaerobic phase for P release that varied in duration (see Table 1), iii) pumping a 2 g N/L sodium nitrate solution for 4.5e5 min, corresponding to 45e50 mg nitrate-N/L in the reactor, iv) an anoxic phase for P uptake and denitrification that varied in duration (see Table 1), v) settling for 30 min, vi) decanting 15 or 10 L of the supernatant for 10 min, and vii) 70 min idle. The durations of the anaerobic and anoxic phases were varied to allow the P release and P uptake reactions enough time to complete (Wu et al., 2013). Synthetic saline sewage was prepared with 20% seawater and 80% freshwater (~0.7% salinity in the mixture) to simulate the influent carbon-to-sulfur (C/S) ratio of typical saline sewage in Hong Kong (approximately 1.0 mg C/mg S). Following Wu et al. (2014), the synthetic sewage comprised 60 mg of NHþ 4 -N/L, 20 mg of PO3 4 -P/L, 267 mg acetate-COD/L, 133 mg propionic-COD/L (400 mg COD/L or 150 mg TOC/L total) and 150e200 mg S/L of sulfate, on average. The reactor was continuously operated for 250 days in four stages: the start-up (Day 1 to Day 45) and stable operation (Day 46 to Day 105) stage (Stage I); the optimized operation stage from Day 106 to Day 125 (Stage II); the failure stage from Day 126 to Day 225 (Stage III), during which different methods were tested to restore the failed system from Day 158 to Day 225; and the system restored stage from Day 226 to Day 250 (Stage IV). Details of the operation in each stage are described as follows: in Stage I, the sludge concentration was kept at 11.8 g/L; in Stage II, a portion of the sludge (approximately 160 g) was removed on Day 105 from the reactor to decrease the sludge concentration to 6.5 g/L; in the initial period of Stage III (Day 126 to Day 157), sludge was removed again on Day 126 to further decrease its concentration to 4.6 g/L to further induce the system into deterioration and failure (disappearance of P removal), otherwise proper sludge reduction continued until failure was achieved. Thereafter, four attempts were made to restore the failed system as follows: in Trial-1 (Day 158 to Day 250) sludge concentration was raised to 7.5 g/L by reducing the effective working volume of the reactor from 30 to 20 L and then adding 10 L of influent instead of 15 L; in Trial-2 (Day 180 to Day 200) salinity was reduced from 0.7 to 0.4% by halving the fraction of seawater used in the synthetic sewage, while extra sodium sulfate solution was added to maintain the same C/S ratio; in Trial-3 (Day 201 to Day 225) the influent COD concentration

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Table 1 Experimental stages, SBR operating conditions and performance (mean ± standard deviation). Experimental stages

SBR operating conditions

Stage

Loading rate (mg COD/g VSS cycle)

Salinity (%)

Cycle length (h)a

Anaerobic phase Time (h)

Cin./Sin. (mg C/mg S)

Time (h)

NO 3

26.3 ± 0.3 50.0 ± 0.5

0.7 0.7

62 / 31 22

24 / 9 8

0.48 ± 0.03 0.47 ± 0.04

36 / 20 12

50 ± 5 45 ± 5

0.7 0.7 0.4 0.7 0.7

13 13 13 13 14

5 5 5 5 6

0.45 0.50 0.49 1.02 0.50

6 6 6 6 6

45 45 45 45 50

Stage I Stage II Stage III Failure Trial-1 Trial-2 Trial-3 Stage IV

Days

105 20 32 22 21 25 25

62.5 37.7 35.7 83.8 37.8

± ± ± ± ±

1.2 7.7 0.6 1.5 3.3

Reactor performance Anoxic phase b

± ± ± ± ±

0.05 0.03 0.01 0.010 0.05

dose (mg N/L)c

± ± ± ± ±

MLSS (g/L)

MLVSS (g/L)

SRT (d)

ORP (mV, SHE)

11.8 ± 0.5 6.5 ± 0.4

7.6 ± 0.4 4.0 ± 0.2

92 ± 10 85 ± 8

300 to 100 300 to 100

± ± ± ± ±

-d -d -d -d 65 ± 12

300 300 300 300 300

5 5 5 5 5

4.6 7.0 7.5 7.8 7.3

± ± ± ± ±

0.3 0.3 0.3 0.3 0.2

3.2 5.3 5.6 6.4 5.3

0.2 0.5 0.5 0.5 0.5

to to to to to

100 100 100 50 80

a

The average cycle length in different stages; the cycle length gradually decreased in Stage I and was determined for achieving complete P-release and P-uptake reactions. The initial ratio of TOC to sulfate-sulfur (Cin./Sin.) was calculated from the initial concentration of total carbon divided by the initial concentration of sulfate-sulfur in reactor at the beginning of anaerobic phase. Initial sulfate-sulfur concentration in reactor was monitored throughout the operation (data not shown). c The initial concentration of nitrate at the beginning of the anoxic phase was calculated theoretically and specified in terms of the entire reactor liquid volume. d No data available. b

was doubled. Finally in Stage IV (Day 226 to Day 250), based on the results of Stage IeIII, sodium sulfide solution (5 g S/L) was added to the reactor to set the sulfide concentration to 25 ± 5 mg S2/L prior to the anoxic phase in selected cycles (see details in Section 3.1.1). No sludge was purposely wasted in each operation stage although small quantities were removed for the deterioration tests as described above, samples during typical cyclic tests and routine sampling, and due to some suspended solids that escaped via the effluent. The sludge retention time (SRT) was determined based on sludge wastage to be 92, 85 and 65 days for Stages I, II, and IV respectively, except Stage III (Table 1). 2.2. Monitoring of reactor performance Four to six mixed liquor samples of 2e4 mL were periodically taken from the reactor in both P release and P uptake phases in each cycle for monitoring the reactor performance throughout the study. These samples were first filtered with 0.22 mm filters for analyzing volatile fatty acids (VFAs) (e.g. acetate and propionate), total organic carbon (TOC), ortho-P (PO3 4 -P), sulfate, thiosulfate, sulfide, nitrate, and nitrite. Cyclic behavior in the reactor was examined in cyclic tests at approximately 20-day intervals. In each test, 12e24 samples of 20 mL were collected from the reactor, of which 2 mL were used for measuring the bulk liquid components and the remaining 18 mL were used for analyzing bacterial internal polymers (i.e. PHA including poly-b-hydroxybutyrate (PHB), poly-bhydroxyvalerate (PHV), poly-b-hydroxy-2-methylvalerate (PH2MV), glycogen, and poly-S2-/S0). At the end of each test, another 10 mL sample was collected for analyzing total phosphorus (TP), mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS). 2.3. Analytical methods MLSS, MLVSS and sulfide were determined according to the Standard Methods (APHA, 2005). Key anions in the bulk liquid, including acetate, propionate, chloride, nitrite, nitrate, phosphorus, sulfate and thiosulfate were determined by ion chromatography (Shimadzu Prominence Liquid Chromatograph). TOC was analyzed using a TOC analyzer (Shimadzu TOC-L). PHA was determined by gas chromatographyemass spectrometry (GC-MS) (Agilent 7890A5975C) according to Oehmen et al. (2005a). Glycogen was analyzed by the anthrone method according to Jenkins et al. (2004). Poly-S2-/

S0 were determined by the sulfite method (Jiang et al., 2009). The analytical methods for PHA, glycogen, and poly-S2-/S0 are described in Wu et al. (2013). 2.4. Microbial community analysis In order to explore the roles of functional bacteria in the DSEBPR system, diversity of the microbial communities was revealed by 16S rRNA gene analysis as described in Hao et al. (2015). Sludge was sampled from the reactor on Days 60, 120, 145 and 245, representing the typical sludge at each operational stage. The whole process includes DNA extraction, polymerase chain reaction (PCR) amplification, pyrosequencing and data analysis. Supporting information and results of the bacterial analysis are provided in SI 2. 3. Results and discussion 3.1. System operation from stable state towards failure followed by restoration 3.1.1. Long-term reactor performance Table 1 summarizes the key operating parameters as mentioned in Section 2.1. Fig. 1 shows the changes in TOC removal, P release, P uptake, sulfate reduction and sulfate production over time. During the start-up period (Day 1 to Day 45) in Stage I, TOC removal efficiency increased from 49 to 93%, corresponding to an increase in sulfate reduction from 21.5 to 59.3 mg S/L and P removal efficiency from 27.2 to 68.4%. From Day 46 to Day 105 (stable operation period) in Stage I, the system maintained a relatively stable performance, with 94% of TOC removed, 64.5 mg S/L sulfate reduced in the anaerobic phase and 58.0 mg S/L of sulfate produced with almost all nitrate consumed in the anoxic phase. However, P removal fluctuated at an average 45.0%, though both P release and P uptake increased to 15.4 and 21.0 mg P/L, respectively (see Fig. 1). The sludge concentration in Stage I (11.8 g/L) was close to that of a typical anaerobic system (10e80 g/L, Lettinga et al., 1993), but much higher than that of a conventional EBPR system (4e5 g/L, Oehmen et al., 2005b; Carvalho et al., 2007). Such a high sludge concentration might induce acidogensis of substrates into VFAs under anaerobic conditions (Ruel et al., 2002), which produced 10e20 mg C/L of extra acetate in some cycles in Stage I. This was intensified when the cycle length was 31e62 h as required to complete organics and nitrate removal (see Table 1), and caused

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Fig. 1. Operational parameters and performance variations during the 250eday study period: (a) Biomass concentration ( ); (b) initial TOC concentration in the reactor (C), final anaerobic TOC concentration (A), anaerobic TOC removal efficiency (-); (c) P release ( ), P uptake ( ), P removal efficiency (A); and (d) sulfate reduction ( ), sulfate production ( ).

sulfate reduction to rise up to 78 mg S/L on Day 76. Such a high sulfate reduction (>60 mg S/L) could disrupt or inhibit the phosphorus removal activity of an EBPR system (Yamamoto-Ikemoto et al., 1991; Baetens, 2001; Saad et al., 2015). Hence, the high sludge concentration of 11.8 g/L in this stage might explain the unstable and lower phosphorus removal during the stable

operation period. In order to control the sulfate reduction, the sludge concentration was reduced to 6.5 g/L by removing sludge at the beginning of Stage II (on Day 106). The sulfate reduction decreased by 80.4% and the phosphorus removal efficiency was enhanced by 58.6% (see Fig. 1b and c). Meanwhile, the cycle length of the reactor was correspondingly reduced to 22 h due to increased bacterial activity. These results confirm that sludge concentration significantly affects the sulfur conversion and P removal efficiency. In the initial period of Stage III (from deterioration to failure, Day 126 to Day 157), the sludge concentration was further decreased to 4.6 g/L. This operation successfully initiated a gradual system failure, as shown by the decrease in both sulfur conversion (reduction and production) and phosphorus removal (Fig. 1). Both the sulfate reduction and sulfate production decreased rapidly and almost ceased on Day 140. Similarly, the P removal performance decreased to only 11.9%. Corresponding changes in internal polymers and microbial communities are discussed in Section 3.2 and Section 3.3 respectively. In conjunction with system failure, the sludge color changed from black to yellow (see Fig. S5, SI 3), possibly due to a decrease in metals (e.g. ferrous) and sulfide in the sludge (Fuhs and Chen, 1975) reflected by the decreased sulfate reduction from Stage I to Stage III. Starting from Day 158 in Stage III, three methods were first attempted to rescue the failed system. The methods involved: 1) increasing the sludge concentration (Trial-1), 2) lowering the salinity (Trial-2), and 3) doubling the influent COD concentration (Trial-3). All these attempts failed to recover the system and in some cases even worsened the P removal (see Fig. 1bec). The fourth attempt, i.e. adding 25 ± 5 mg S2/L of sulfide into the reactor prior to the anoxic phase, was made in Stage IV. In this investigation, sulfide was added in each cycle from Day 226 to Day 238 for 13 consecutive days. As a result, the P uptake quickly recovered and the P removal efficiency reverted to 92.1%. In order to verify its effectiveness, sulfide addition was stopped from Day 239 to Day 244 for 6 days, during which the P removal efficiency rapidly decreased to 31.5% (see Fig. 1c). Sulfide addition was resumed from Day 245 to Day 250 for 6 days, causing P removal to return to earlier levels as shown in Fig. 1c. Powder X-ray diffraction (XRD) analysis results of the sludge sample taken on Day 245 revealed it was not chemical P removal since no P-containing crystals formed in the reactor despite the sulfide addition (Fig. S6, SI 4). In addition to enhanced P removal, the sulfate reduction also recovered partly to 8.5 mg S/L in Stage IV. Repeated experiments confirmed that anoxic addition of external sulfide to DS-EBPR could effectively restore the failed system. The shifting of reactor performance (e.g. P removal) from stable state to failure followed by restoration correlated well with the variations in P content in the sludge (P/VSS) (see Fig. 1 and Fig. 2b). During stable and optimized operation periods (Stages IeII), the P/ VSS value increased from approximately 27 to 63 mg P/g VSS over 125 days, but it decreased significantly by 74.6% when the system failed in Stage III, and finally rose back to 27 mg P/g VSS when the system was restored in Stage IV. Moreover, the changes in P content in sludge were also consistent with the variations in MLVSS/MLSS ratios (Wang et al., 2013), as shown in Fig. 2c. 3.1.2. Cyclic performance Four cyclic tests (C1 (Day 73), C2 (Day 114), C3 (Day 176), and C4 (Day 238)) in each stage were selected to display cyclic profiles under different operating conditions (see Fig. 3). As shown in Fig. 3aeb, the biological conversions in terms of sulfur conversion, P release, P uptake, denitrification and formation of internal polymers clearly displayed a stable DS-EBPR phenotype in C1 and C2, whereas C3 showed a failed system (Fig. 3c). Specifically, the

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glycogen content of sludge in C3 increased by approximately 60%, which likely indicates GAOs enrichment in the reactor (Oehmen et al., 2005b). In C4, the 25 ± 5 mg S/L of external sulfide added was gradually consumed within 6 h in the anoxic phase. Meanwhile poly-S2-/S0 had built up initially but was subsequently consumed for P uptake, resulting in 95.2% of P removal (see Fig. 3d). The formation of internal polymers changed significantly in all four tests, correlating well with the system performance (see Fig. 3), which will be discussed in Section 3.2.1. The results obtained in Section 3.1 reveal a relationship between sulfur conversion and biological phosphorus removal in the DSEBPR system, confirming a similar phenomenon in marine sediments (Brock, 2011) and some activated sludge plants treating sulfate-laden wastewaters (Ginestet et al., 2015; Daigger et al., 2015). 3.2. Effect of sulfur conversion on internal polymer conversions and phosphorus removal 3.2.1. Effect of sulfur conversion on internal polymer conversions The anaerobic internal polymers changed in PHA synthesis, glycogen hydrolysis, and poly-S2-/S0 production in each cyclic test as well as in the long-term poly-S2-/S0 accumulation in sludge, as shown in Fig. 4. The average stoichiometric ratios are summarized in Table 2 to illustrate the effect of sulfur conversion on the conversion of internal polymers.

Fig. 2. P content in sludge and variations in sludge concentration: (a) ratio of P release to VFA uptake (þ); (b) ratio of total P to VSS ( ); (c) ratio of MLVSS to MLSS ( ).

293

In Stages IeII, the anaerobic sulfate reduction remained in a range of 25e78 mg S/L, theoretically consuming 50e156 mg/L of COD, corresponding to PHA synthesis and glycogen hydrolysis (per C mmol VFA uptake) of 0.35e0.42 mmol PHA-C/mmol C and 0.27e0.41 mmol Gly-C/mmol C, respectively (Fig. 4a). In parallel, 86.0% of poly-S2-/S0 reverted to sulfate, leaving the remaining poly-S2-/S0 in the sludge to grow from 25.2 to 61.6 mg S/g VSS (Fig. 4b). The phosphorus removal varied between 45.0 and 84.2% during this period (Fig. 1b). In contrast, when sulfate reduction decreased to almost zero during system deterioration (Day 114 to Day 157, in Stage III) (see Fig. 1d), PHA synthesis and glycogen hydrolysis increased to 1.21 mmol PHA-C/mmol C and 0.92 mmol Gly-C/mmol C respectively (Fig. 4a), causing a drop in poly-S2-/S0 accumulation to 13.5 mg S/gVSS and in the phosphorus removal efficiency to 11.9% (Fig. 1b). Moreover, the ratios of PHV/PHA surged from approximately 5% in Stage I to 42e67% in Stage III. As reported by Oehmen et al. (2005b) and Acevedo et al. (2012), under the same VFAs feed, proliferation of GAOs exhibits higher PHV production and more glycogen hydrolysis in the anaerobic phase. Hence, the weakened sulfate reduction caused by a lower sludge concentration in the reactor significantly affected the formation of internal polymers, which might have further triggered a dynamic shift from a stable DS-EBPR performance to a GAOs-enriched state. This was further supported by comparing the anaerobic stoichiometric values obtained in the present study with previous metabolic model predictions and/or experimental results for conventional PAOs and GAOs mechanism pathways as well as a deteriorated DS-EBPR system (see Table 2). From Day 158 to Day 225 in Stage III, when Trials 1e3 were conducted, these profiles remained similar to that of the failed state, e.g. 0.8 mg S/L of sulfate reduction, 1.2e1.4 mmol PHA-C/ mmol C of PHA synthesis, 0.6e0.9 mmol Gly-C/mmol C of glycogen hydrolysis, and 14.2 mg S/g VSS of poly-S2-/S0 accumulation, corresponding to a low P removal of 23.1%. During the restoration (Stage IV), adding external sulfide enabled sulfate reduction to partly recover (8.5 mg S/L), while PHA synthesis and glycogen hydrolysis decreased by approximately 30% correspondingly (Fig. 4a). Meanwhile, poly-S2-/S0 accumulation increased slightly to 27.2 mg S/g VSS, thereby returning P removal to an average 78.1% (Fig. 1c and Fig. 4b). All of the above results indicate that sulfur conversion has significant effects on the formation of internal polymers, especially those polymers serving as energy sources for P uptake. The conversion will undoubtedly affect the final P removal in a DS-EBPR system, as will be further discussed in the next section. 3.2.2. Correlation of sulfur conversion with phosphorus removal The correlation of sulfur conversion with biological phosphorus removal is established by curve-fitting as shown in Fig. 4c. The data were collected from Day 51 to Day 125 and from Day 157 to Day 177, representative of the typical reactor performance under stable, deteriorated and failure operation. Additionally, poly-S2-/S0 almost accumulated continuously during these days eliminating its effect on P removal (see Fig. 4b). The results show that the DS-EBPR system could achieve a good P removal performance (>80%) with a critical sulfate reduction range of 15e40 mg S/L. Beyond this range, e.g. <15 or >40 mg S/L, anaerobic sulfate reduction was not in favor of P removal (<60%). In conformity with this hypothesis, the electron flow based on different sulfate reductions was further estimated according to Wu et al. (2013) as detailed in SI 5. Maintaining a moderate level of sulfate reduction subsequently induced appropriate levels of PHA and poly-S2-/S0 production during the anaerobic phase (see Table 2), possibly driving functional bacteria to produce more

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Fig. 3. Cyclic changes of the key compounds in the system measured in the cyclic tests: (a)e(d) shows the cyclic tests C1 (Day 73), C2 (Day 114), C3 (Day 176) and C4 (Day 238), respectively. Acetate ( ), propionate ( ), PHB ( ), PHV ( ), PHA ( ), glycogen ( ), phosphorus ( ), sulfate ( ), poly-S2-/S0 ( ) and nitrate ( ).

energy for bacterial growth, maintenance, and synthesis of poly-P than that of other conditions (see Table 3). This energy-based estimation has further proved that an appropriate level of sulfate reduction is crucial for biological phosphate removal in this EBPR system.

3.3. Functional bacteria and mechanism 3.3.1. Variations in the microbial community The 16S rRNA gene analysis identified potential functional bacteria in the DS-EBPR system, as shown in Fig. 5. After filtering and trimming adapters, barcodes and primers for quality, the

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observed with changes in reactor performance from stable state to failure (see Fig. 5bed) and confirmed by Venn diagram (Fig. S3 in SI 2): Under stable operation (Stage I), SRB (including Desulfobacter (16.9%), Desulfobulbus (4.7%), Desulfuromonas (2.6%), etc.) made up 33.4% of the Day 60 sample whereas SOB (including Sedimenticola (9.0%), Thiohalomonas (8.0%), Thiotrichaceae (3.3%), Thiobacillus (1.0%), etc.) made up 23.5% (see Fig. 5b). In addition, a few fermenters (e.g. BD2-2_norank) were found in the sludge (5.2%), further supporting acidogenesis causing surplus sulfate reduction in this stage (as mentioned in Section 3.1.1). In the Day 120 sample taken during the optimized stage (Stage II), known fermenters disappeared (see Fig. 5a) and the populations of SRB and SOB decreased to 10.7% and 8.3% respectively. At the same time, small proportions of methanogens (2.2%), denitrifiers (10.3%), and GAOs (1.6%, e.g. Candidatus Competibacter) appeared (Fig. 5c). In the Day 145 sample taken during the period of failure (Stage III), SRB and SOB both disappeared almost entirely, but the population of GAOs increased significantly to 60.6%, dominating the bacteria population. In addition, the sample consisted of 3.8% methanogens and 5.1% denitrifiers (Fig. 5a and d). When SRB and SOB both dominated the reactor sludge, the system was in a stable condition. When sulfur conversion was weak, GAOs out-competed sulfur conversion bacteria (e.g. SRB, SOB) leading to a failed system. Although the competition between GAOs and conventional PAOs has been broadly studied in the past decades (Oehmen et al., 2007; Lopez-Vazquez et al., 2009), the competition between sulfur conversion bacteria and GAOs deserves further study. In Stage IV, the populations of SRB and SOB recovered by 4.7% and 2.6% respectively (Fig. 5e) through the addition of external sulfide, even though the GAOs population remained at 54.1% and explained the gradual but consistent P recovery observed in Stage IV. As GAOs are a highly competitive species, their reduction is expected to be a gradual process (Oehmen et al., 2007). The family Rhodobacteraceae (classified into the ‘others’ group) accounted for 18.1%, 13.1%, 4.1% and 1.5% of all bacteria in Stages I, II, III, and IV, respectively (Fig. 5a). These trends are similar to those displayed by the SRB and SOB populations. Rhodobacteraceae are reported to be closely involved in sulfur and carbon biogeochemical cycling in anaerobic environments (Pujalte et al., 2014), a further study of their roles in the DS-EBPR system is scheduled.

Fig. 4. Anaerobic internal polymer variations, poly-S2-/S0 accumulation in sludge and the relationship between sulfate reduction and P removal efficiency (aec): a) Anaerobic internal polymer variations (PHB ( ), PHV ( ), PHA ( ), glycogen ( ), sulfate reduction ( )); b) poly-S2-/S0 accumulation in sludge ( ); and c) the relationship between sulfate reduction and P removal efficiency ( ).

samples were left with 7700, 6600, 8558, and 8000 sequences (Fig. S2 in SI 2). The diversity of the microbial communities was well represented by the sequencing depth of the four samples, with rarefaction curves approaching plateaus (Fig. S2 in SI 2). The GOOD's coverages (Table S1 in SI 2) were 99%, 98%, 98% and 99% with 3% dissimilarity for these four samples. Fig. 5a shows the abundances of the bacterial community at the genus level during different stages. Similar to the results of Wu et al. (2014), the conventional PAOs, e.g. Accumulibacteria and Tetrasphaera, were not detected in these four samples (further verified by fluorescence in-situ hybridization (FISH) analysis as shown in Fig. S4). But obvious shifts in the microbial community were

3.3.2. Bacterial roles in the DS-EBPR system Recently, sulfur conversion bacteria, especially SOB, have been reported to play a crucial role in the precipitation of phosphorus in marine sediments (Schulz and Schulz, 2005; Arning et al., 2009; Brock, 2011; Lepland et al., 2014; Alsenz et al., 2015). Schulz and Schulz (2005) found a correlation between the occurrence of SOB (i.e. Thiomargarita in Namibian shelf sediments) and increased deposition of phosphate. Holmkvist et al. (2010) found that Thioploca influenced the mineralization of P in shelf sediments from central Chile. Brock (2011) reported that Beggiatoa released phosphorus from internally stored polyphosphate (polyP) in pulses creating steep peaks of phosphorus in marine sediments, thereby inducing the precipitation of phosphorus-rich minerals. Alsenz et al. (2015) reported geochemical evidence for the link between sulfur conversion and phosphorus accumulation in a Late Cretaceous upwelling system. Although some SOB species, such as Thiomargarita, Thioploca and Beggiatoa, could accumulate and deposit phosphorus, these species were seldom found in our DS-EBPR system. Several observations indicate sulfur conversion bacteria (SRB and SOB) can synergistically

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Table 2 Comparison of anaerobic carbon transformations, sulfur conversion and P release with available literature studies and metabolic model predictions using acetate and propionate as carbon sources. Study

Carbon source

P/VFAa

Poly-S/VFAb

Gly/VFAc

PHA/VFAc

PHB/VFAc

PHV/VFAc

PH2MV/VFAc

Stage I Stage II Stage III Stage IV Sulfur cycle-associated EBPR Wu et al. (2013) Wu et al. (2014) DPAO metabolic models Kuba et al. (1996) Experimental studies DPAO Carvalho et al. (2007) Carvalho et al. (2007) Experimental studies DGAO Zeng et al. (2003a) PAO metabolic models Smolder et al. (1994) Oehmen et al. (2005b) GAO metabolic models Zeng et al. (2003b) Oehmen et al. (2005b)

HAc/Pro HAc/Pro HAc/Pro HAc/Pro

0.20 0.30 0.05 0.11

0.33 0.16 0.01 0.08

0.27 0.31 0.81 0.71

0.38 0.64 1.24 0.89

0.36 0.42 0.49 0.38

0.02 0.22 0.75 0.50

0 0 0 0

HAc HAc

0.29 0.24

0.18 0.16

0.06 0.60

0.31 0.58

0.31 0.58

0 0

0 0

HAc

0.36

0

0.50

1.33

1.33

0

0

HAc Pro

0.16 0.40

0 0

0.50 0.32

1.37 0.97

1.10 0

0.27 0.40

0 0.57

HAc

0

0

1.15

1.87

1.40

0.47

0

HAc Pro

0.50 0.34

0 0

0.50 0.33

1.33 1.23

1.33 0

0 0.56

0 0.67

HAc Pro

0 0

0 0

1.12 0.70

1.86 1.83

1.36 0.13

0.46 0.71

0 0.99

DPAO means denitrifying polyphosphate accumulating organisms; DGAO means denitrifying glycogen-accumulating organisms. Stoichiometric ratios were used as the average values in Stages IeIV. a Unit is P mmol C mmol1. b Unit is S mmol C mmol1. c Unit is C mmol C mmol1.

Table 3 Energy calculation for different sulfate reduction conditions. Sulfate Specific e storage reduction Poly-S (mg S//l)

a

Energy consumed for growth

Energy consumed for Energy remaining for glycogen production polyP synthesis

PHB ATP generated Yield coefficienta Biomass produced YATPb ATPGrowth ATP Glycogen (mmol/l) (mmol ATP/l) (g VSS/g COD) (mg VSS/l cycle) (mmolATP/g VSS) (mmolATP/l) (mmolATP/l)

Low <15 7.8 Moderate 15e40 4.0e7.8 High >40, mean 56 2.38 b

Energy budget Biomass growth

<30.3 19.9e30.3 16.0

0.21 0.21 0.21

0.04 0.04 0.04

95 95 95

3.8 3.8 3.8

21.1 8.1e21.1 7.0

ATP polyP (mmolATP/l) <5.4 5.4e8.0 5.2

Coefficient of YATP was obtained from Stephanopoulos et al., Metabolic Engineering: Principles and Methodologies, p 71, (1998). True yield coefficient was determined following the thermodynamic energetic calculation method (Henze et al., 2008).

dominate P release and P uptake functions in this system: 1) keeping the anaerobic sulfate reduction induced by SRB to a moderate level played a key role in achieving good DS-EBPR performance; 2) when SOB oxidized an appropriate level of internal polymers formed in the anaerobic phase, sufficient energy could be generated for P removal; 3) when SRB and SOB dominated in this reactor, the system remained stable, but when SRB and SOB populations decreased rapidly, biological P removal deteriorated; 4) the restoration of SRB and SOB activities through the addition of sulfide was accompanied by a recovery in P removal. Based on the above results and discussion, the potential functional bacteria and a possible process metabolism of DS-EBPR has been illustrated (see Fig. 6). Under anaerobic conditions, carbon sources are taken up by both SRB and SOB for sulfate reduction to form poly-S2-/S0 in SRB and synthesize PHA in SOB respectively. At the same time, SOB perform P release and glycogen degradation. Subsequently under anoxic conditions, SOB oxidize intracellular PHA and poly-S2-/S0 by using nitrate as the electron acceptor for P uptake, glycogen replenishment and sulfate production. In this microecological system, SRB and SOB work together to produce a synergic effect on P release and P uptake. When compared to the metabolism of conventional EBPR, SOB in the DS-EBPR system seemingly replace the role of conventional PAOs by generating sulfur and polyP pools to perform sulfur conversion-associated

phosphorus removal, as well as compete with GAOs for limited carbon sources. Despite strong support of the above mentioned mechanism from results on reactor performance changes, internal polymer conversions, associated estimated energy yields, and microbial community shifts, the full metabolic pathways of SRB and SOB are yet to be revealed. The main problems related to the storage of polyP and the pathways of sulfur conversion as proposed in Fig. 6 are: 1) Which specific SOB perform P uptake and store it as polyP? 2) What is the composition of poly-S2-/S0? 3) Is poly-S2-/S0 stored intracellularly or extracellularly in SRB/SOB? 4) Is poly-S2-/S0 transferred from SRB to SOB and if so how? Further study to address these questions is deemed necessary. 4. Conclusions This study systematically investigated the functional bacteria and the possible mechanism of DS-EBPR by manipulating the deterioration, failure and restoration of this system through changing sludge concentration. Throughout the entire operation, reactor performance, anaerobic stoichiometry, sulfur conversion, and diversity of the microbial communities were monitored. Four methods were tested to recover the deteriorated DS-EBPR system, including: 1) increasing the sludge concentration, 2) lowering the

G. Guo et al. / Water Research 95 (2016) 289e299

297

Fig. 5. (a) Taxonomic classification of bacterial 16s rDNA reads retrieved from Day 60, 120, 145 and 245 samples at the genus level using RDP classifier with a confidence threshold of 80%; (bee) distribution of bacterial community on Days 60, 120, 145 and 245, respectively. Sulfate-reducing bacteria ( ), sulfide-oxidizing bacteria ( ), glycogen-accumulating organisms ( ), methanogens ( ), denitrifiers ( ), other bacteria ( ).

salinity, 3) doubling the carbon sources, and 4) adding sulfide. The main conclusions are summarized as follows: 1. Keeping a moderate sludge concentration (~6.5 g/L) is conducive to stable DS-EBPR performance.

2. A synergistic relationship exists between anaerobic sulfate reduction and biological phosphorus removal, viz. maximum P removal could be achieved at the appropriate values of 15e40 mg S/L sulfate reduced.

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Fig. 6. Schematic illustration of the roles of functional bacteria in the DS-EBPR system (modified from Wu et al. (2014) and Alsenz et al. (2015)).

3. SRB and SOB participate synergistically in biological phosphorus removal in the DS-EBPR system. 4. GAOs may be enriched in the DS-EBPR system under reduced sludge concentrations and may out-compete SRB and SOB, thereby causing DS-EBPR deterioration. 5. The DS-EBPR performance can recover through the addition of 25 ± 5 mg/L of sulfide in the anoxic phase. Acknowledgments The research was partly supported by the Hong Kong Research Grants Council (grant no. 16213515) and Project 2011 under the jurisdiction of the Beijing Municipality. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2016.03.013. References Acevedo, B., Oehmen, A., Carvalho, G., 2012. Metabolic shift of polyphosphateaccumulating organisms with different levels of polyphosphate storage. Water Res. 46 (6), 1889e1900. Alsenz, H., Illner, P., Ashckenazi-Polivoda, S., Meilijson, A., 2015. Geochemical evidence for the link between sulfate reduction, sulfide oxidation and phosphate accumulation in a late cretaceous upwelling system. Geochem. Trans. 16, 2. APHA, 2005. Standard Methods for the Examination of Water and Wastewater, twenty-first ed. American Public Health Association (APHA)/American Water Works Association (AWWA)/Water Environmental Federation (WEF), Washington DC, USA. Arning, E.T., Birgel, D., Brunner, B., Peckmann, J., 2009. Bacterial formation of phosphatic laminites off Peru. Geobiology 7 (3), 295e307. Baetens, D., 2001. Enhanced Biological Phosphorus Removal: Modeling and Experimental Design. Ph.D. Dissertation. Ghent University, Belgium. Bassin, J.P., Kleerebezem, R., Dezotti, M., van Loosdrecht, M.C.M., 2012. Simultaneous nitrogen and phosphate removal in aerobic granular sludge reactors operated at different temperatures. Water Res. 46 (12), 3805e3816. Beer, J., 1999. Seawater Intrusion in Coastal Aquifers-Concepts, Methods and Practices. Kluwer Academic Publishers, The Netherlands. Brock, J., 2011. Impact of Sulfide-oxidizing Bacteria on the Phosphorus Cycle in Marine Sediments. Ph.D. Dissertation. University of Bremen, Bremen. Carvalho, G., Lemos, P.C., Oehmen, A., Reis, M.A.M., 2007. Denitrifying phosphorus

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