Impact of Cr(VI) on P removal performance in enhanced biological phosphorus removal (EBPR) system based on the anaerobic and aerobic metabolism

Bioresource Technology 121 (2012) 379–385

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Impact of Cr(VI) on P removal performance in enhanced biological phosphorus removal (EBPR) system based on the anaerobic and aerobic metabolism Jing Fang, Pei-de Sun ⇑, Shao-juan Xu, Tao Luo, Ju-qing Lou, Jing-yi Han, Ying-qi Song School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012, China

h i g h l i g h t s " Cr(VI) had a significant toxity effect on EBPR system. " PAOs were more sensitive to Cr(VI) than GAOs and the other bacteria were. " P removal performance in EBPR system was able to recover after 5 mg L

a r t i c l e

i n f o

Article history: Received 7 March 2012 Received in revised form 7 June 2012 Accepted 2 July 2012 Available online 10 July 2012 Keywords: Cr(VI) Enzymes Poly-b-hydroxyalkanoates (PHA) Glycogen Recovery treatment

1

Cr(VI) shock.

a b s t r a c t Influence of Cr(VI) on P removal in enhanced biological phosphorus removal (EBPR) system was investigated with respect to the composition of poly-phosphate-accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), the transformation of poly-b-hydroxyalkanoates (PHA) and glycogen, enzymes’ activities, and the intracellular Cr. Whether EBPR system could revive after Cr(VI) shock was also explored. Results showed P removal performance was completely inhibited by Cr(VI) with the concentration more than 5 mg L 1. PAOs were more sensitive to Cr(VI) than GAOs and the other bacteria were. PHA consumption, glycogen synthesis and adenylate kinase’s activity had been inhibited by 5 mg L 1 Cr(VI). Both adenylate kinase’s activity and P removal efficiency were negatively correlated with the intracellular Cr. Recovery experiments revealed that P removal performance with 5 mg L 1 Cr(VI) shock could revive after a 2-day recovery treatment, while systems with high level Cr(VI) (20 and 60 mg L 1) shock could not. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Enhanced biological phosphorus removal (EBPR) is a widely implemented process in wastewater treatment plants to remove phosphorus from wastewater (Oehmen et al., 2007). Successful operation of EBPR depends on numerous process operational factors, especially the quality of wastewater. Although manufacturers of China are obliged to treat their wastewater containing large amounts of heavy metals on site in their factories, some still discharge their untreated wastewater into public wastewater treatment plant where the wastewater treatment system was then shocked. Shock loads of heavy metals often result in the loss of microbial viability, community structures and finally reduction of nutrient treatment efficiency or even failure of biological processes (Madoni et al., 1996; Hu et al., 2004). The influence of heavy metals on the conventionally activated sludge system and nitrification performance has been thoroughly studied (You et al., 2009; Cecen et al., 2010; Stasinakis et al., ⇑ Corresponding author. Tel.: +86 571 88905799; fax: +86 571 88832369. E-mail address: [email protected] (P.-d. Sun). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.07.001

2002; Chen and Gu, 2005). Once enter cells, heavy metal ions can interact with thiol groups and destroy protein’s structures and functions. They can also inhibit enzyme’s activities and thus cease normal metabolism processes (Nies, 1999). The toxicity of heavy metal ions are determined by the species and concentration of heavy metals, pH, MLSS, sludge age, sludge retention time and acclimatization (Rayne et al., 2005; Hu et al., 2003; Wang et al., 2011). Hu et al. (2002) demonstrated that inhibition of nitrification was not a function of the total analytical heavy metal concentration but strongly correlated with free cation concentration. They further reported that nitrification inhibition was not a function of the sorbed metal fraction but correlated well with intracellular heavy metal fractions (Hu et al., 2003). Currently the effect of heavy metals on the EBPR process has rarely been studied. Rayne et al. (2005) firstly reported that the P removal efficiency decreased dramatically when tin (Sn) levels in the solid exceeded 4 lg L 1. Recently, Wang et al. (2011) investigated the shock load effect of copper on the behavior of poly-phosphate-accumulating organisms (PAOs) in EBPR systems. This research showed that the P removal was not adversely affected by spiking with 2 mg L 1 Cu2+, but deteriorated completely after

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a Cu2+ shock at the concentration of 4 mg L 1. They also found that the inhibitory effect of Cu was reduced by increasing pH. Meanwhile, Tsai and Chen (2011) explored the influence of sludge retention time on tolerance of Cu invasion for EBPR system. They pointed out that the effect of Cu on EBPR was complicated and varies with mechanisms. However, previous studies have hardly focused on the effect of heavy metals on the composition of bacterial population and the activity of enzymes in the EBPR process. In EBPR system, one known competibacter as glycogen accumulating organisms (GAOs) has been frequently found (Oehmen et al., 2007; Lu et al., 2006). GAOs compete with PAOs for the often limited carbon sources, without performing P removal. Whether heavy metals have the same inhibitory-toxic effect on PAOs and GAOs remains uninvestigated. Additionally, most metabolic processes of microorganisms in EBPR system are related to enzyme catalysis. The alkaline phosphatase and adenylate kinase are identified to be important enzymes related to the process of phosphate removal in EBPR (Oehmen et al., 2007). Phosphatase catalyzes the hydrolysis of terminal phosphate of polyphosphate. Adenylate kinase is related to the degradation of poly-P in anaerobic process and adenosine triphosphate (ATP) synthesis. Chromium (Cr) is considered as toxic to aquatic environment and often encountered in the sewage released from the effluent of steelworks, chromium electroplating, leather tanning and chemical manufacturing (Orozco et al., 2008; Vaiopoulou and Gikas, 2012). Cr(VI) is highly soluble and can be transported into cells, which is more toxic than Cr(III) (Stasinakis et al., 2003a). Therefore, the objectives of this study were to (i) investigate the acute toxic effect of Cr(VI) on P removal performance in EBPR system based on the anaerobic and aerobic metabolism of the biomass; (ii) to evaluate the relationships between the inhibition of P removal, the enzymes activities of alkaline phosphatase and adenylate kinase, and the intracellular Cr of the biomass; (iii) to explore whether P removal performance in EBPR system can revive after Cr(VI) shock. Wide Cr(VI) concentrations (5–60 mg L 1) were selected in this study in order to test the tolerance of EBPR system on Cr(VI). 2. Methods 2.1. Obtaining the EBPR biomass of PAOs from the activated sludge Two laboratory scale (working volume was 10 L) anaerobic– aerobic sequencing batch reactors (SBR) were used for the enrichment of PAOs. The seed sludge for PAOs acclimation experiment came from the return sludge sewage in Qige Wastewater Treatment Plant, Hangzhou, China. The SBR reactor was operated for four cycles per day. Each cycle lasted for 6 h, involving 5 min of feeding, 2.5 h of anaerobic stirring, 3 h of aerobic reaction, 15 min of precipitation, 5 min of effluent discharge, and 5 min of idling. Hydraulic retention time (HRT) was 24 h and sludge retention time (SRT) was 8 d. Sludge concentration was about 2500 mg L 1, and the temperature of the activated sludge units was kept at 20 ± 2 °C using an air conditioning. The pH value in both reactors was maintained at 7.5–8.0 using 0.1 M HCl or 0.1 M NaOH, and dissolved oxygen (DO) level in aerobic reactors was 2–3 mg L 1. According to Lu et al. (2006), the sole carbon source in the synthetic feed was alternated between acetate and propionate with a time interval of 8 d, in order to provide selective advantages to PAOs over GAOs. The EBPR biomass was acclimated for more than 75 days. 2.2. Synthetic wastewater The influent solution to the reactors consisted of solution A, solution B and trace element solution (Table 1). Every 2.5 L influent

contains 0.370 solution A, 2.125 L solution B and 5 mL trace element solution in each cycle of reactor operation. The initial aqueous COD and phosphate concentration of each cycle in the reactor were about 200 and 10 mg L 1, respectively. 2.3. Batch experiments with EBPR sludge Batch tests were conducted in 2 L jacketed magnetically stirred glass reaction vessels. The sludge for the batch tests was taken from the final aerobic compartment of the SBR system on Day 95. The EBPR sludge from two SBR reactors were firstly mixed and then washed for three times with distilled water, after which they were transferred into the reaction vessels. The incubation procedures were conducted according to Section 2.1, and the pH value of reactors was also maintained at 7.5–8.0 using 0.1 M HCl or 0.1 M NaOH. Sodium acetate was used as the sole carbon source during batch experiments. 2.3.1. Batch Cr(VI) toxicity experiments at different Cr(VI) concentrations The washed EBPR sludge was resuspended with the synthesis water to a volume of 20 L, from which 16 L sludge was evenly divided into eight batch reactors. Sludge concentration in each reactor was 2500 mg L 1. A required amount of potassium dichromate solution was added to the batch reactors at the beginning of cycle to provide a constant Cr(VI) concentration of 5, 20 and 60 mg L 1 for each treatment cycle, respectively. Each Cr(VI) concentration was set in two parallel and the reactors with no Cr(VI) were used as the control. At the beginning of the anaerobic stage of each cycle, acetate and KH2PO4 were quickly added into each batch reactor to reach initial COD concentration of 200 mg L 1 and PO43 –P concentration of 10 mg L 1. The added amount of acetate and KH2PO4 for each cycle was calculated according to the residual P and acetate concentrations in the liquid at the end of previous cycle. This toxicity experiment lasted for 24 h (4 cycles). In order to provide insight into the toxicity mechanisms of Cr(VI) on EBPR system, the activities of enzymes (the alkaline phosphatase and adenylate kinase), the degradation and production of PHA and glycogen of EBPR biomass and the intracellular Cr concentrations were monitored in 5 mg L 1 Cr(VI) treatment reactors. The compositions of the bacterial population were also monitored at the initial state and the end of 5 mg L 1 Cr(VI) treatment. 2.3.2. The recovery experiments for EBPR system after Cr(VI) shock At the end of the Cr(VI) toxicity experiments, the EBPR sludge in each batch reactor (except for the two control reactors) was firstly washed with distilled water for three times to remove the Cr(VI) residue. Subsequently, the recovery experiment was conducted with no Cr added into the EBPR system. The recovery experiment lasted for 2 days (7 cycles). In the recovery experiment of 5 mg L 1 Cr(VI) treatment reactors, the activities of enzymes, intracellular Cr concentrations, the content of PHA and glycogen as well as the microbial population were monitored. 2.4. Analytical methods The liquid samples were immediately filtered through qualitative filter paper for analysis of PO43 –P and COD. The PO43 –P, COD, MLSS and VSS were analyzed in accordance with standard methods (APHA, 1998). Poly-b-hydroxyalkanoates (PHA) analysis was performed using the method of Oehmen et al. (2005) to determine poly-b-hydroxybutyrate (PHB), poly-b-hydroxyvalerate (PHV), and poly-b-hydroxy-2methylvalerate (PH2MV). Glycogen was determined as detailed in Bond et al. (1999). The activity of alkaline phosphatase and adenylate kinase were measured accord-

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J. Fang et al. / Bioresource Technology 121 (2012) 379–385 Table 1 The composition of synthetic wastewater (g L

1

).

Solution A

Solution B

NH4Cl MgSO4
7H2O CaCl2
2H2O Peptone Yeast extract powder Sodium acetate/propionate ATU

1.02 1.2 0.19 0.01 0.01 10/5.6(ml) 0.008

Trace element solution

KH2PO4 K2HPO4.3H2O

ing to the methods described by Goel et al. (1998) and van Groenestijn et al. (1989), respectively. For bacterial quantification, fluorescent in situ hybridization (FISH) was performed as described in Amann (1995). The EUBMIX was used to target the entire bacterial community (Daims et al., 1999). The PAOMIX probe (PAO462, PAO651 and PAO846) (Crocetti et al., 2000) was used to target PAOs. The GAOMIX probe (GAOQ431 and GAOQ989) (Crocetti et al., 2002) in conjunction with the GBG2 probe (Kong et al., 2002) were used to target GAOs. For image acquisition, an epifluorescence microscope (IX71, Olympus Corp., Tokyo, Japan) was used. Quantification of the PAOs and GAOs communities with respect to the entire bacterial population was determined via FISH image analysis (Crocetti et al., 2002). The final quantification result was expressed as a mean percentage obtained from 30 images. Intracellular Cr concentrations were measured with a modified EDTA washing procedure (Hu et al., 2003). Briefly, suspensions of the PAOs enrichment culture with and without Cr were centrifuged at 3000 rpm for 5 min. Microbial cell pellets retained after centrifugation were resuspended in 30 mL of washing solution (1 mM EDTA, pH 7.0, and 0.1 M NaCl to prevent osmotic shock) and agitated at 150 rpm for 30 min to remove surface-bound metal, followed by further centrifugation at 3000 rpm for5 min. Such wash procedure was conducted for three times. Then the supernatant was removed and 5 mL of 4 M nitric acid was added to the pellets, followed by totally transfering the contents to a glass reaction tube. The suspension containing 4 M nitric acid was digested at 100 °C for 24 h. The cooled digest was filtered (0.45 lm), and the Cr concentration in the filtrate was measured by atomic absorption (AA) spectrometry. 2.5. Statistical analysis Statistical analyses were performed using SPSS 11.5 for Windows (SPSS Inc., USA). Pearson correlation coefficients were determined by correlating activities of enzymes with P removal efficiency and with intracellular Cr of the EBPR biomass. The significance probability levels of the results were given at the p < 0.01(⁄⁄). 3. Results and discussion The initial composition of the microbial population in EBPR system, characterized by using FISH, is shown in Table 2. PAOs was the

0.103 0.173

FeCl3
6H2O H3BO3 CuSO4
5H2O KI MnCl2
4H2O Na2MoO
2H2O ZnSO4
7H2O CoCl2
6H2O EDTA

1.5 0.15 0.03 0.18 0.12 0.06 0.12 0.15 10.0

predominant bacteria in the sludge before Cr(VI) treatment experiment, indicating that the EBPR biomass was successfully enriched. 3.1. Effect of Cr(VI) on P and COD removal in EBPR system Three Cr(VI) concentrations (5, 20, 60 mg L 1) were selected according to the tolerance of the PAOs to Cr(VI), in comparison with the control test. The effects of various concentrations of Cr(VI) on P and COD removal are shown in Fig. 1. During continuous operation for four cycles, the P removal efficiencies in the EBPR system were significantly decreased with increased Cr(VI) (Fig. 1a). The P removal efficiency had decreased to 0 just after 1 cycle loading for EBPR system with 60 mg L 1 of Cr(VI), indicating that 60 mg L 1 Cr(VI) had already exceeded the tolerance limit of the PAOs biomass. Similarly, P removal performance was completely inhibited after two cycles when treating with 20 mg L 1 Cr (VI). For the case of 5 mg L 1 Cr(VI) treatment, P removal performance gradually decreased and finally became invalid after three treatment cycles. The influence of Cr(VI) on P release and uptake rate in EBPR system was also monitored. As shown in Fig. 2, at the anaerobic stage, the P release rate diminished gradually with treatment cycles and completely ceased at the 4th cycle with 60 mg L 1 Cr(VI) loading. Similarly, much less P release rate was also found for the case of 20 mg L 1 Cr(VI) loading after three treatment cycles compared to its initial state, though P release maintained at the normal level in the first two cycles. Conversely, for the case of 5 mg L 1 Cr(VI) loading, the P release rate was much higher than that of its initial state. At the aerobic stage, the P uptake rate was significantly inhibited by all Cr(VI) loadings (Fig. 2b). For the case of 60 mg L 1, no P uptake was found in EBPR system just after 1 treatment cycle. Similarly, the P uptake was completely inhibited after two treatment cycles for the case of 20 mg L 1. For the case of 5 mg L 1 Cr (VI), P uptake rate began to decrease after three treatment cycles and decreased to 48% of its initial P uptake level at the 4th treatment cycle. From the above-mentioned results, it is not difficult to conclude that the process of P uptake in EBPR at the aerobic stage was more sensitive to Cr(VI) than P release at the anaerobic stage. Take the case of 20 mg L 1 Cr(VI) for example, P release rate was almost normal at the 2nd treatment cycle, however, the P uptake rate had been completely suppressed at this time and thus led to the failure of P removal performance in EBPR system. The COD removal performance of EBPR system was also significantly influenced by Cr(VI), except for the case of 5 mg L 1 Cr(VI) loading (Fig. 1b). In the case of 60 mg L 1 Cr(VI), the COD removal

Table 2 The relative abundance of PAOs, GAOs and the other bacteria with respect to the total cell stained. Stage

Operation cycles

PAOs

GAOs

The other bacteria

Initial End of 5 mg L Recovery

0 4 11

+++ ++ +++

+ ++ +

+ ++ ++

1

Cr(VI) treatment

+++ = High abundance; ++ = discrete abundance; + = minimal abundance.

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(a)

(b) 100 toxication stage

80

60

COD removal efficiency %

P removal efficiency %

100

Control -1 Cr(VI) 5 mg L -1 Cr(VI) 20 mg L -1 Cr(VI) 60 mg L

40

20

recovery stage

toxication stage

80

Control -1 Cr(VI) 5 mg L -1 Cr(VI) 20 mg L -1 Cr(VI) 60 mg L

60 40 recovery stage

20 0

0

0

2

4

6

8

10

0

12

2

4

6

8

10

12

Treatment Cycles

Treatment Cycles

Fig. 1. Variations in phosphate (a) and COD (b) removal efficiencies in batch reactors with different Cr(VI) treatments during operation cycles.

P release rate in anaerobic -1 -1 (mg P g VSS hr )

Control -1 Cr(VI) 5 mg L -1 Cr(VI) 20 mg L -1 Cr(VI) 60 mg L

toxication stage

16 14 12 10 8 6

recovery recoverystage stage

4 2

(b)

16 14

P uptake rate in aerobic -1 -1 (mg P g VSS hr )

(a)

18

Control -1 Cr(VI) 5 mg L -1 Cr(VI) 20 mg L -1 Cr(VI) 60 mg L

toxication stage

12 10 8 6 4

recovery stage

2 0

0 0

2

4

6

8

10

12

Treatment Cycles

0

2

4

6

8

10

12

Treatment Cycles

Fig. 2. The rate of P release (a) and uptake (b) in batch reactors under different Cr(VI) treatments during operation cycles.

efficiency reduced to zero after two treatment cycles. While for the case of 20 mg L 1 Cr(VI), the COD removal efficiency constantly decreased and finally reduced to 14% after four treatment cycles. It should be pointed out that, for the cases of 20 and 60 mg L 1 Cr(VI), the apparent characters of sludge significantly changed at the 4th treatment cycle, which became white, semitransparent and bulking with large amounts of foam floating on the liquid surface. At the end of the 4th treatment cycle, the sludge aggregates were markedly broken down and the effluent was quite muddy, indicating the loss of sludge during drainage process. This suggested that the bacteria were deeply poisoned, which were consistent with previous studies. Once the concentration of heavy metals exceeded the tolerance of microorganism, they greatly inhibited the growth of activated sludge or even led to bacterial demise, which all resulted in the decrease of COD removal (Stasinakis et al., 2002). In our experiment, the COD removal just referred to the removal of volatile fatty acids (VFAs) such as acetate. As we know, during the anaerobic period, PAOs in EBPR system mainly take up VFAs to store them in the form of PHA (Oehmen et al., 2005). However, the other bacteria like GAOs and heterotrophic bacteria which often presented in EBPR system can also take up VFAs to support their growth. As shown in Table 2, at the end of 5 mg L 1 Cr(VI) treatment (the end of 4th cycle), results of FISH analysis revealed that the PAOs abundance decreased significantly compared to their initial stage, while the abundance of GAOs and other heterotrophic bacteria increased. This may demonstrate that PAOs were much more sensitive to Cr(VI) than GAOs and other bacteria. As a result, Cr(VI) had larger toxic effect on P removal than that on COD

removal in EBPR system. The changes in microbial community structure caused by exposure to high metal contamination were reported in literature data (Principi et al., 2006; Stasinakis et al., 2003b; Turpeinen et al., 2004). 3.2. Effect of Cr(VI) on the synthesis and consumption of PHA and glycogen by EBPR biomass Cr(VI) concentration of 5 mg L 1 was chosen to study the influence mechanism of Cr(VI) on EBPR system with respect to the activities of enzymes, the transformation of PHA and glycogen, and the intracellular Cr accumulation in the biomass. PHA is a crucial compound in PAOs metabolism. During the anaerobic period, the consumption of polyphosphate and glycogen supplied energy and/or reducing power for PHA formation. In the subsequent aerobic stage, PAOs used the stored PHA as the carbon and energy sources for biomass growth, glycogen replenishment and orthophosphate uptake and storage as poly-P (Mino et al., 1998; Lu et al., 2006). The degraded and produced level of PHA in the presence of 5 mg L 1 Cr(VI) are described in Fig. 3. After three treatment cycles, the production of PHA was comparable with those of the control, however, the consumption of PHA in the biomass were generally lower than those of the control. This result was consistent with the corresponding P release and uptake process. The release of P was stimulated by 5 mg L 1 Cr(VI) treatment, which supplied the enough energy for transformation of PHA, while P uptake at the aerobic stage was inhibited because of the shortage of energy

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J. Fang et al. / Bioresource Technology 121 (2012) 379–385

(a) control

100

PHA produced PHA degraded

PHA produced PHA degraded

90

PHA produced and degrated -1 (mg gVSS )

90

PHA produced and degrated -1 (mg gVSS )

(b) Cr(VI) load

100

80 70 60 50 40 30 20 10 0

80

toxication stage

70

recovery stage

60 50 40 30 20 10 0

0

1

2

3

4

5

6

7

8

9

10 11 12

0

1

2

3

4

Treatment Cycles 1

Fig. 3. Effect of 5 mg L

7

8

9

10 11 12

3.3. Effect of Cr(VI) on the activities of alkaline phosphatase and adenylate kinase The activities of alkaline phosphatase and adenylate kinase during 5 mg L 1 Cr(VI) treatment were presented in Fig. 5. As shown in Fig. 5a, alkaline phosphatase’s activity was found to increase during the exposure to Cr(VI). Such stimulated activity of alkaline phosphatase had been reported by many previous studies. Xie et al. (2010) reported that Mn2+, Co2+, Pb2+ and Cr6+ ions stimulated the activities of alkaline phosphatase in activated sludge system. Vinodhini and Narayanan (2008) also found the alkaline phosphatase activities were stimulated by heavy metals in experimental fish. Alkaline phosphatase is an important enzyme in immune defense, which gives a good picture of the general metabolic condition of the organisms (Xiao et al., 2002). The increased activity of alkaline phosphatase has been attributed to an adaptive response in mitigating the Cr toxicity or a stress response of living cells. In this study, such stimulated activity of alkaline phosphatase, which catalyzed the hydrolysis of monophosphate esters, agreed well with the increase of P release at anaerobic stage in EBPR system (shown in Fig. 2). Contrary to the stimulation of alkaline phosphatase activity, the activity of adenylate kinase showed a significant decline in this study (Fig. 5b). The inhibition of adenylate kinase by Cr(VI) increased steadily with treatment cycles. Adenylate kinase is a phosphotransferase enzyme that catalyzes the interconversion of ATP, ADP and AMP, and plays an important role in cellular energy homeostasis. The heavy metal intoxication possibly altered the ATP:ADP equilibrium concentrations for adenylate kinase (Morris et al., 2005). The decrease of adenylate kinase activity with the presence of Cr(VI) was possibly responsible for the loss of P

(a) control

0.036

glycogen degraded glycogen produced

Glyc-degrated and produced -1 (C mg g VSS )

0.030

Glyc-degrated and produced (C mg g VSS-1)

6

Cr(VI) on the transformation of PHA during operation cycles.

provided by PHA degradation. Similar toxic effect of heavy metal on PHA transformation was also reported by Wang et al. (2011). They found that the inhibition of aerobic PHA degradation in response to Cu was greater than the inhibition of anaerobic PHA synthesis in EBPR system. At the end of Cr(VI) treatment (the 4th cycle), both the production and consumption of PHA were significantly decreased. This corresponded with the decreased population of PAOs at this time (Table 2). The decreasing PAOs biomass and PHA consumption did not provide enough energy to uptake P, leading to the failure of P removal in EBPR system. The degraded and produced level of glycogen in the presence of 5 mg L 1 Cr(VI) are shown in Fig. 4. After three treatment cycles, the production of glycogen was completely inhibited though the consumption of glycogen maintained. The persistent degradation of glycogen provided the energy for the production of PHA at anaerobic stage. The zero synthesis of glycogen in EBPR system suggested that the metabolism process of glycogen synthesis was significantly restrained by 5 mg L 1 Cr(VI). Coats et al. (2011) pointed out that the shortage of PHA supply may cause the failure of glycogen synthesis aerobically. PAOs used the stored PHA as the carbon and energy sources for glycogen replenishment at aerobic stage. The PHA degradation and glycogen synthesis had very similar transformation trends under heavy metal stress (Wang et al., 2011). The regulation of glycogen synthesis is quite complex, which requires an activated form of glucose and several enzymes (Berg et al., 2002). Although the PHA production at the 3rd cycle was still almost the same with the control one, the synthesis of glycogen was completely inhibited. We speculate that the other factors that influence glycogen synthesis i.e. the glycogen synthase, might be inhibited. However, the effect of Cr(VI) on glycogen metabolism need to systematically investigate in the future.

0.036

5

Treatment Cycles

0.024 0.018 0.012 0.006

(b) Cr(VI) load glycogen degraded glycogen produced

0.030 toxication stage

0.024

recovery stage

0.018 0.012 0.006 0.000

0.000 0

1

2

3

4

5

6

7

8

Treatment Cycles Fig. 4. Effect of 5 mg L

1

9

10 11 12

0

1

2

3

4

5

6

7

8

Treatment Cycles Cr(VI) on the transformation of glycogen during operation cycles.

9

10 11 12

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J. Fang et al. / Bioresource Technology 121 (2012) 379–385

Control -1 Cr(VI) 5 mg L

150 120 90 60 toxication stage

30

(b)

0.14

Adenylate kinase activity -1 -1 (U g VSS h )

-1

(a)

-1

Alkaline phosphatase activity

(µg p-Nitrophenol g VSS h )

180

Control -1 Cr(VI) 5 mg L

0.12 0.10 0.08 0.06 0.04

toxication stage

0.02

recovery stage

0

0.00 0

2

4

6

8

10

12

0

2

Treatment Cycles

-1

Intracellular Cr concentration mg g

1.2 1.0 0.8 0.6 0.4

toxication stage

recovery stage

0.0 0

2

4

6

8

10

12

Treatment Cycles Fig. 6. The accumulation of Cr in the biomass during operation cycles.

removal efficiency. This would be further approved by the well correlation between P removal efficiency and adenylate kinase activity (see the following section).

3.4. P removal inhibition related to intracellular Cr and enzymes’ activities in EBPR system Metal partitioning by microorganisms generally included extracellular sorption, transmembrane transport and intracellular accumulation when exposed in metal contaminated environment. Hu et al. (2003) found that the inhibition of activated sludge performance was not a function of the sorbed metal fraction but correlated well with intracellular metal fraction. Therefore, the intracellular accumulated Cr in the biomass was determined through the treatment cycles in this study. As shown in Fig. 6, within the four Cr(VI)

Table 3 The correlations between enzymes activities, P removal efficiency and intracellular Cr concentrations in the biomass during 5 mg L 1 Cr(VI) treatment. Pearson correlation coefficient Intracellular Cr concentrations Alkaline phosphatase activity Adenylate kinase activity **

P removal efficiency 0.944**

Intracellular Cr concentrations –

0.537

0.506

0.966**

0.992**

indicate significant correlations at the probability level of p < 0.01.

4

6

8

10

12

Treatment Cycles

Fig. 5. The activities of alkaline phosphatase (a) and adenylate kinase (b) in batch reactors with 5 mg L the active unit.

0.2

recovery stage

1

Cr(VI) treatments during the toxicity and recovery experiment. U is

treatment cycles, the intracellular concentration of Cr gradually increased with treatment time and the maximally accumulated Cr was 1.09 mg g 1 MLSS. Correlation analyses found that both P removal efficiency (r = 0.944, p < 0.01) and the activity of adenylate kinase (r = 0.992, p < 0.01) negatively correlated well with the intracellular Cr (Table 3), suggesting the intracellular Cr was directly responsible for the observed inhibitory effect on P removal process. In order to understand the role of enzymes on P removal, the correlation analysis was also conducted between the activities of enzymes and the P removal efficiency. From Table 3, activity of adenylate kinase was significantly correlated to the P removal efficiency (r = 0.966, p < 0.01), but alkaline phosphatase activity was not. It indicated that adenylate kinase was an important enzyme directly responsible for the P removal process. This result was consistent with the previous study, which indicated that the decreasing activity of adenylate kinase would lead to the lower P metabolism in activated sludge (van Groenestijn et al., 1989). 3.5. Recovery of P removal performance in EBPR system after Cr(VI) shock The recovery experiment was conducted with no Cr addition in the EBPR system, lasting for 2 days. Results showed that the P removal efficiency in EBPR systems kept on 0 after 20 mg L 1 and 60 mg L 1 Cr(VI) shock during 2 days recovery treatment (Fig. 1). Meanwhile, the COD removal efficiency during recovery stage was 0 for the case of 60 mg L 1 Cr(VI) shock and was less than 10% for the case of 20 mg L 1 Cr(VI) shock. This demonstrated that EBPR systems under these two concentrations had been completely destroyed and cannot revive in 2 days. The failure of recovery may be ascribed to the loss of sludge or PAOs population. On the one hand, the loss of sludge occurred at the end of toxic experiment as described in previous section. On the other hand, PAOs showed the most sensitive to Cr(VI), the abundance of which decreased significantly under 5 mg L 1 Cr(VI). The stress of high Cr(VI) concentration was expected to cause more decrease of PAOs population. In EBPR system with 5 mg L 1 Cr(VI) shock, the P removal efficiency had recovered to more than 90% (the normal high P removal). Meanwhile, the degraded and produced level of PHA and glycogen had returned to the normal at the later recovery stage (as shown in Figs. 3 and 4). The activities of alkaline phosphatase and adenylate kinase in EBPR system treated with 5 mg L 1 Cr(VI) shock were also able to recover to the normal level 2 days later, compared with the control (Fig. 5). FISH result demonstrated that the population of PAOs had increased to high abundance of the entire bacterial population, indicating that PAOs were enriched in EBPR system again (Table 2). These results were all in accordance well with the recovery of P removal performance.

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It should be noted that the intracellular Cr of the biomass was decreasing within the recovery treatment cycles (Fig. 6). After seven recovery cycles, the accumulated Cr in the biomass was reduced from 1.09 to 0.57 mg g 1 dry sludge. Such release of Cr by the biomass was possibly responsible for the recovery of enzymes and the P removal processes. Above all showed that EBPR system was able to revive from the shock of 5 mg L 1 Cr(VI) and maintained the stabilization of P removal performance after a short-term recovery treatment.

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