Evaluation of the efficacy and regulation measures of the anammox process under salty conditions

Separation and Purification Technology 132 (2014) 584–592

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Evaluation of the efficacy and regulation measures of the anammox process under salty conditions Hui Chen a, Chun Ma a, Yu-Xin Ji a, Wei-Min Ni a, Ren-Cun Jin a,b,⇑ a b

Department of Environmental Science and Engineering, Hangzhou Normal University, Hangzhou 310036, China Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou 310036, China

a r t i c l e

i n f o

Article history: Received 5 May 2014 Received in revised form 5 June 2014 Accepted 7 June 2014 Available online 14 June 2014 Keywords: Anammox Salinity Adaptation strategy Extra biomass addition Glycine betaine addition

a b s t r a c t The present study aimed to achieve a better understanding of the performances of anaerobic ammonium oxidation (anammox) reactors using different strategies to manage high salt conditions. Batch assays were conducted to ascertain the effects of ion species, and the results demonstrated the different effects on the specific anammox activity. Osmotic pressure was another determining factor with a 50% inhibitory level of 1.4 MPa. Self-adaptation strategies were conducted by running R1 (gradual adaptation) and R2 (rapid adaptation), and the nitrogen removal rates were 3.71 ± 0.63 kg N m3 d1 (R1) and 3.48 ± 0.51 kg N m3 d1 (R2) below 15 g NaCl L1. Additionally, the NRR decreased significantly when the salinity was increased (more severe in R2). Furthermore, the enhanced adaptation tests confirmed that extra biomass addition (EBA) in R2 was effective to resist the salinity stress, whereas the effectiveness of glycine betaine addition (GBA) in R3 was dubious. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Excess nitrogen in wastewater is a significant issue worldwide due to its ability to harm the eco-system and humans. Industrial wastewater from seafood processing, textile dyeing, the chemical, pharmaceutical and petroleum industries, oil and gas production, tanneries, livestock and landfill leachate contains large amounts of ammonium and salts [1–3]. There is no doubt that the effluent discharged should be pre-treated to prevent the contamination of water body. Anaerobic ammonium oxidation (anammox), first discovered in a denitrifying fluidized bed reactor in 1995, is a new process for treating wastewater [4]. In the anammox process, ammonium is oxidized, with nitrite serving as an electron acceptor under anaerobic conditions, producing dinitrogen gas (Eq. (1)) [5]. Today, the anammox process is successfully and extensively applied as a high efficiency and low-cost nitrogen removal technique worldwide [6–10].

NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:256NO3 þ 0:066CH2 O0:5 N0:15 þ 2:03H2 O

ð1Þ

⇑ Corresponding author at: Department of Environmental Science and Engineering, Hangzhou Normal University, Hangzhou 310036, China. Tel.: +86 571 88062061; fax: +86 571 28865333. E-mail address: [email protected] (R.-C. Jin). http://dx.doi.org/10.1016/j.seppur.2014.06.012 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.

To date, several efforts have been made to determine the influence of salinity on the anammox process [2,3,11], and Soto et al. [12] indicated that in the methanogenic system, moderate concentrations of positive ions such as Na+, K+ and Mg2+ could stimulate microbial growth, whereas excessive amounts restricted the growth and even higher concentrations resulted in substantial inhibition or toxicity. Hitherto, majority of the researchers have considered the influence of NaCl on the anammox process, but little work has been performed to explore other types of salts. Moreover, Chen et al. [13] concluded that the cation predominantly determined the toxicity of the salt rather than the anion, and they reviewed the toxic actions of light and heavy metal ions, and the studies about the impact of negative ions on the anammox process still remain scarce. Kartal et al. [3] conducted a study investigating the adaptation of a freshwater anammox population to high salinity wastewater. They reported that the anammox bacteria from a freshwater system successfully adapted to salt concentrations that gradually increased to as high as 30 g L1. Bassin et al. [1] explored the effect of different salt acclimatization strategies on the microbial diversity, activity and settling properties of a nitrifying sludge. Nevertheless, very few similar investigations about optimization of adaptation strategies of anammox bacteria to wastewater containing salinity have been performed. High salinity results in high osmotic pressure (OP), and microorganisms in high salinity die or become plasmolyzed and dormant. One way for the cells to survive the osmotic stress is to

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accumulate intracellular organic solutes called ‘‘compatible solutes’’. These solutes can be synthesized by the cell or provided by the medium. Glycine betaine is one of the solutes studied in the methanogenic system [14]. However, the benefit of glycine betaine in a high-salinity anammox process remains a mystery. The addition of biocatalysts is another effective way to conquer the effects of high salinity. As reported previously, the nature of the substrate (ammonium and nitrite), the organic compound composition and the presence of toxicants such as antibiotics can destroy the anammox system. If the anammox biomass were inhibited by these factors, achieving the recovery of the SAA from deterioration can be difficult. An effective alternative is to add some fresh anammox sludge with high activity, which results in an increased quantity of bacteria in the system. As a result, the addition of a biocatalyst can help the anammox system to resist negative external factors. Tang et al. [15] claimed that biocatalyst (anammox biomass) addition was effective for the treatment of pharmaceutical wastewater in the anammox reactor due to the expected resistance of the highly enriched anammox granules to toxic substances. It is worth considering biocatalyst addition as a strategy to address high salinity in the anammox reactor. Therefore, the objectives of this study are to determine the influence of ion species on the anammox process and to evaluate the differences between acclimation strategies. Regulation measures, such as extra biomass addition (EBA) and glycine betaine addition (GBA), were taken to confirm their ability to relieve the inhibition caused by the salinity. 2. Materials and methods 2.1. The anammox reactors Three upflow anaerobic sludge blanket (UASB) reactors with working volumes of 1 L were used for the enrichment and cultivation of the anammox bacteria. The hydraulic retention time (HRT) of each reactor was kept constant at 2 h, and the reactors were placed in a thermostatic room at 35 ± 1 °C and covered with a black cloth to prevent light inhibition. The reactors were fed synthetic wastewater via three peristaltic pumps at the bottom of the reactor. The nitrogen gas produced was released from an outlet on the top of each reactor. There was a water outlet port in the middle of the reactor from which samples were taken.

ion species on the anammox process in serum bottles and the total volume of each bottle was 140 mL with a liquid phase volume of 120 mL. (NH4)2SO4 and NaNO2 were supplied as the sources of NH+4-N and NO 2 -N, respectively. The initial concentrations of the 1 NH+4-N and NO , and the pH was 2 -N were set at 100 mg L adjusted to 7.4–7.6 with 1 mol L1 hydrochloric acid or sodium hydroxide. The bottles were flushed with pure argon gas (99.99%) and firmly closed with 4-mm-thick butyl rubber septa to maintain an anaerobic environment. Finally, the bottles were placed in a constant temperature vibrator at 35 ± 1 °C and on a shaker at 180 rpm. The ammonium and nitrite concentrations were analyzed at regular intervals using syringe filters to take samples from the bottles. In the study of the short-term effects, the sludge was pre-processed with the same method used to determine the SAA, and the anammox granules used for the study of the short-term effects were collected from a steadily operated UASB reactor with a maximum SAA of 14.13 mg N g1 VSS h1 (VSS represents volatile suspended solid). Different salts with a series of dosages were added into the bottles to confirm the short-term effects (Table 2). Eventually, the SAA was calculated by the equation SAA = MSCR/ VSS (MSCR is the maximum substrate consumption rate). 2.5. Analytical methods and calculations During the initial phase, the concentrations of NH+4-N, NO 2 -N and NO 3 -N and the pH of each reactor were determined once a day, and the detection frequency decreased to once every two days in the mid and late phases. The measurements of suspended solid  (SS), VSS, NH+4-N, NO 2 -N, NO3 -N and the pH were performed according to Standard Methods [18]. The inhibition was described as the relative anammox activity ratio (RSAA) (%).

RSAA ¼ ½SAAinhibition =SAAreference   100

ð2Þ

where SAAinhibition is the SAA of the anammox bacteria at a corresponding inhibition level and SAAreference is the SAA of the anammox bacteria without inhibition. The OP was calculated using the Donnan equilibrium ion distribution and the OP equations [19]. The modified non-competitive inhibition model (Eq. (3)) was applied to reveal the inhibitory characteristics of osmotic stress on anammox activity.

2.2. Inoculum and synthetic wastewater

I ð%Þ ¼ 100  1 

1

!

1 þ ð½OP=aÞb

ð3Þ

The seeding sludge was taken from an anammox reactor in the laboratory that had run steadily for more than half a year. Ammonium and nitrite were added to the mineral medium, as required, in the forms of (NH4)2SO4 and NaNO2, respectively. The mineral medium was prepared according to Yang and Jin [16].

where I (%) is the inhibition response, a is the 50% inhibitory OP (MPa) and b is the fitting parameter.

2.3. Operation strategies

3.1. The short-term effects of salt with different ion species

Three reactors named R1, R2 and R3 ran for a total of 163 days. R1 and R2 were run with different NaCl supply strategies and R2 was also used to test the feasibility of EBA, while R3 was used to confirm the effectiveness of GBA. The operational conditions of the reactors are listed in Table 1.

The effects of the presence of different salts (NaCl, KCl and Na2SO4) at several concentrations on the SAA are listed in Table 2. As presented in Table 2, when the concentration of Na+ was 0.25 mol L1, RSAA was 57.6%; when the concentration of Na+ increased to 0.5 mol L1, RSAA decreased to 19.0%. For K+, RSAA had a similar tendency with values of 55.5% and 15.5% when the concentrations of K+ were 0.25 and 0.5 mol L1, respectively. K+ probably inhibited the bioactivity more severely than did Na+ at the same molar concentration. Additionally, when the concentration + of Na+ was 0.25 mol L1, the conversion of NO 2 -N to NH4-N (RS) was 1.36, which approached the theoretical stoichiometric ratio of the anammox reaction [5]. With an increase of the Na+

2.4. Specific anammox activity (SAA) batch assays Batch assays to determine the specific anammox activity (SAA) were performed according to the methodology described previously [17], based on the concentrations of NH+4-N and NO 2 -N over time. The batch assays were also conducted to confirm the effect of

3. Results and discussion

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Table 1 The operational conditions of R1, R2 and R3. Reactor

Strategy

ISC (g L1)

Days

R1

Gradual adaptation

5 10 15 20 25 30

1–6 7–16 17–30 31–69 70–85 86–163

R2

Rapid adaptation

Extra biomass addition (10 mL anammox sludge per day)

10 20 30 30

1–38 39–74 75–155 156–163

Glycine betaine addition (0.05–5 mmol L1)

5–30

1–163

R3 ISC: influent salt concentration.

Table 2 The effects of NaCl, KCl and Na2SO4 on the specific anammox activity (SAA) observed in batch assays. Salt type

Salt concentration (mol L1)

NH+4-N removal rate (mg N g1 VSS h1)

NO 2 -N removal rate (mg N g1 VSS h1)

Osmotic pressure (MPa)

SAA (mg N g1 VSS h1)

RSAA (%)

I (%)

Control NaCl

0 0.25 0.5 0.25 0.5 0.25 0.5

6.72 3.44 1.06 0.84 1.55 2.32 0.19

7.38 4.70 1.65 3.40 0.64 0.06 0.55

0 1.33 2.61 1.33 2.61 1.97 3.89

14.13 8.14 2.71 7.78 2.19 2.68 0.75

100 57.6 19.2 55.1 15.5 19.0 5.3

0 42.4 80.8 44.9 84.5 81.0 94.7

KCl Na2SO4

concentration to 0.5 mol L1, the ratio increased to 1.55, divergent from the theoretical stoichiometric ratio. In comparison, the ratios were 1.92 and 0.41 when the concentration of K+ was 0.25 and 0.5 mol L1, respectively. Based on the values of the stoichiometric ratio, it can be proposed that with the increase in ion concentration, anammox was more significantly affected by the K+ concentration than by the Na+ concentration.  The RSAA for SO2 4 or Cl presented a negative correlation with the ion concentration. When the concentration of SO2 and Cl 4 was 0.25 mol L1, the RSAA values were 19% and 57.6%, respectively. RSAA fixed at 5.3% and 19.2% when the ion concentrations reached 0.5 mol L1, which might occur because the positive ion concentration of Na2SO4 is twice that of NaCl, whereas the negative ion concentrations are equal. Therefore, during the next stage, the influence of the negative ion concentration was studied while the concentration of the positive ions was kept constant. As shown in Table 2, keeping the Na+ concentration at 0.5 mol L1, when the concentration of Cl was 0.5 mol L1, RSAA was 19.2%, but RSAA was 19.0% when the concentration of SO2 was kept at 4 0.25 mol L1. Mosquera-Corral et al. [20] performed similar experiments regarding partial nitrification in a SHARON reactor in the presence of salts. One result indicated that three salts (NaCl, KCl and Na2SO4) provoked similar effects (40% decreases) on the specific ammonia oxidizing activity at the same molar concentration of 100 mmol L1. Interestingly, in their study, Na2SO4 had the smallest effect, and the other salts performed better than did Na2SO4, which may have occurred because the salt concentrations tested by Mosquera-Corral et al. were much lower than were those in our study. The differences may be further explained considering the distinctions between nitrifying bacteria and anammox bacteria. High nitrogen wastewater also contains large amounts of other ions such as chloride and sulfate. These ions tend to exert a high OP on microorganisms. Generally, a high OP can lead to the dehydration of the cell. To determine the relationship between the SAA and OP, a series of experiments were performed. Based on these results, the SAA decreased obviously with an increase in the OP (Table 2).

The effects of the OP on anammox can be described by the noncompetitive inhibition model, as expressed in Eq. (4). The 50% inhibitory level of OP for the anammox biomass was calculated to be 1.40 MPa.

I ð%Þ ¼ 100  1 

1 1 þ ð½OP=1:40Þ2:92

! ðR2 ¼ 0:9170Þ

ð4Þ

3.2. Self-adaptation strategies 3.2.1. Gradual adaptation According to the performance of R1, in which the influent salt concentration (ISC) increased gradually from 5 to 30 g L1, the test was conducted in six stages (Table 3). Fig. 1A presented the nitrogen loading rate (NLR), nitrogen removal rate (NRR) and ISC during the entire test period. There was no inhibition in R1 when operating with an ISC of 5 g L1. The removal of NH+4-N and NO 2 -N (NRENH and NRENO) were 82% and 95%, respectively. The effluent concentrations of NH+4-N and NO 2 -N were stable, ranking at 34.5 ± 5.0 and 12.0 ± 4.3 mg L1, respectively. After 7 days of stable running, the ISC increased to 10 g L1, which resulted in a rapid increase of the effluent NH+4-N and NO 2 -N concentrations, which reached 58.6 and 18.8 mg L1, respectively. NRENH and NRENO, respectively, rose from 72.8% and 91.9% to 80.2% and 92.8% within the following 10 days. Simultaneously, NRR varied from 4.15 to 4.36 kg N m3 d1 at a similar nitrogen load rate (NLR). As a result, the ISC was changed to 15 g L1, and the effluent NO 2 -N became 24.6 ± 13.3 mg L1. Based on the results of R1, an ISC of 15 g L1 did not cause long periods of variability in the effluent and NO 2N concentration. After 12 days of operation, the ISC was propelled to 20 g L1, at which the performance of R1 started deteriorating. By day 41, the NRR declined to 0.79 kg N m3 d1, and the maximum effluent NH+4-N and NO 2 -N concentrations were 145.7 and 122.6 mg L1. NRR increased slightly and slowly again in the next days, and during days 66–72, the effluent NH+4-N and NO 2 -N concentrations were stable at levels of 129.8 ± 9.7 and

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H. Chen et al. / Separation and Purification Technology 132 (2014) 584–592 Table 3 The performance of R1 and R2 during the self-adaptation test. Reactor

Stage

Time (d)

Salinity (g L1 NaCl)

NLR (kg N m3 d1)

NRR (kg N m3 d1)

Eff. NH+4-N (mg L1)

Eff. NO 2 -N (mg L1)

NH+4-N removal efficiency (%)

NO 2 -N removal efficiency (%)

R1

P11 P12 P13 P14 P15 P16

1–6 7–16 17–30 31–72 73–85 86–163

5 10 15 20 25 30

5.42 ± 1.01 5.08 ± 0.56 4.86 ± 0.87 4.76 ± 0.48 5.10 ± 0.88 4.42 ± 0.47

4.48 ± 0.99 4.03 ± 0.58 3.66 ± 0.73 2.33 ± 0.70 1.77 ± 0.98 1.53 ± 0.46

34.7 ± 5.0 41.4 ± 11.2 50.0 ± 27.0 94.0 ± 27.7 138.8 ± 12.9 124.4 ± 33.1

12.0 ± 4.3 16.0 ± 6.7 24.6 ± 13.3 76.0 ± 21.0 138.8 ± 12.9 84.8 ± 20.0

84.7 ± 4.9 80.9 ± 5.3 73.3 ± 12.9 52.1 ± 12.7 34.4 ± 9.9 37.7 ± 12.8

94.2 ± 1.9 92.3 ± 2.9 87.6 ± 5.9 59.9 ± 12.1 46.6 ± 12.7 49.8 ± 10.3

R2

P21 P22 P23

1–37 38–73 74–155

10 20 30

4.92 ± 0.75 4.79 ± 0.28 4.50 ± 0.73

3.71 ± 0.63 2.85 ± 0.67 1.16 ± 0.45

37.8 ± 17.6 70.6 ± 28.8 144.6 ± 31.9

21.5 ± 10.0 53.7 ± 22.4 108.5 ± 13.4

77.7 ± 15.2 64.9 ± 13.4 30.1 ± 10.0

89.2 ± 5.0 71.8 ± 12.8 40.1 ± 7.5

Eff.: effluent. NLR: nitrogen loading rate. NRR: nitrogen removal rate.

Fig. 1. The evolution of the NLR and NRR values of R1 (A) and R2 (B) during the self-adaptation tests.

45.9 ± 2.6 mg L1. The ISC increased to 25 g L1when the pseudosteady state of the reactor was obtained. The NLR and NRR were 4.76 ± 0.14 and 1.41 ± 0.18 kg N m3 d1, respectively. Finally, the ISC was fixed at 30 g L1 after 13 days of operation, during which R1 was severely inhibited, coinciding with the drop in NRR from 1.59 to 0.97 kg N m3 d1, and the effluent NH+4-N and NO 2 -N concentrations reached top values of 149.2 and 121.0 mg L1, respectively. There was no sign of any improvement in R1 by day 95. As a result, the concentration of the influent substrate was reduced to 182 mg L1 to protect the reactor from the negative consequences of high salinity and NO 2 -N [21]. The capacity of R1 slowly recovered from the inhibition and eventually showed an NRR of 2.14 kg N m3 d1 on day 163. 3.2.2. Rapid adaptation The performance of R2 during the experiment is shown in Fig. 1B. During days 1–37 (P21 in Table 3), the ISC was 10 g L1. At this stage, the NLR and NRR were 4.92 ± 0.75 and 3.71 ± 0.63 kg N m3 d1, respectively. These results indicated that the addition of 10 g L1 NaCl did not affect the capacity of R2 and that the reactor could revive rapidly after slight fluctuations. The effluent NH+4-N and NO 2 -N concentrations remained steady for

3 days, reaching 30.8 ± 2.4 and 25.0 ± 2.1 mg L1 by day 34. Then, the ISC was adjusted to 20 g L1, at which point R2 deteriorated immediately. This adjustment led to a decrease of the NRR from 3.92 to 3.16 kg N m3 d1. Furthermore, the R2 continued to deteriorate with the decline in the NRR to a minimum value of 1.29 kg N m3 d1. The effluent NH+4-N and NO 2 -N concentrations, respectively, increased to 147.7 and 112.7 mg L1 from 32.5 and 26.2 mg L1. The ISC then increased to 30 g L1 on day 75; as a result, the NRR decreased severely, and capacity was lost. The NLR and NRR when ISC was 30 g L1 were 4.56 ± 0.63 and 1.22 ± 0.40 kg N m3 d1, respectively. R2 was run at a pseudo-steady state with an NRR of 3.71 ± 0.63 kg N m3 d1 when the ISC was 10 g L1. The capacity of R2 degraded with the increase in the ISC and when the effluent 1 NH+4-N and NO , while the 2 -N concentrations exceeded 100 mg L 1 ISC was fixed at 30 g L . 3.2.3. Performance comparison of R1 and R2 A comparison between the NRR and effluent NO 2 -N concentration for R1 (gradually acclimated to 30 g NaCl L1) and R2 (rapidly acclimated to 30 g NaCl L1) is presented in Table 3. The shock effects of the ISCs in the NRR and effluent NO 2 -N concentration

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were more pronounced in R2 than in R1. Eventually, R2 was more sensitive to the salt stress of 30 g NaCl L1 (a 76.0% drop in NRR) than R1 (a 69.4% drop in NRR). Because the anammox biomass was sensitive to the salinity shock, an increase in salt elicited a lower NRR. As a result of the larger variations in the salt concentrations in R2, the microorganisms in R2 were much less likely to survive the salinity shocks. Thus, R2 performed at a worse capacity. The results obtained in this study were consistent with the previous studies conducted by Ma et al. [22] and Yu and Jin [23]. These findings indicate that a more gradual increase in salinity was a better choice for the anammox biomass to adapt to stress conditions and display improved performance. 3.2.4. Stoichiometric ratio The stoichiometric ratio can be applied as an index or as evidence indicating the performance of anammox and the evolution of anammox consortia. The theoretical stoichiometric ratio of RS is 1.32, and the ratio of the production of NO 3 -N to the consumption of NH+4-N (RP) was 0.26 [5]. However, under high salt conditions, the ratios of R1 and R2 in Fig. 2 diverged from the theoretical values. RS decreased whereas RP increased with the increase in ISC. For R1, RS was 1.10 ± 0.19, and RP was 0.18 ± 0.05 when the ISC was at 5–15 g L1. Running with an ISC of 20–25 and 30 g L1, the values of RS and RP, respectively, for R1 were 1.13 ± 0.19 and 0.35 ± 0.13 as well as 1.11 ± 0.28 and 0.43 ± 0.19. For R2, during P21 (ISC 10 g L1), RS and RP were 1.18 ± 0.45 and 0.21 ± 0.08, respectively, and the values changed to 1.13 ± 0.19 and 0.38 ± 0.14, respectively, during P22 (ISC 20 g L1) and to 1.08 ± 0.40 and 0.52 ± 0.22, respectively, during P23 (ISC 30 g L1). The stoichiometric ratios were shown to deviate from the theoretical values under a variety of stress conditions [19,24]. The results observed in this work may indicate that the presence of salinity changes the stoichiometric ratios for the anammox reaction due to the inhibition of anammox bacteria [25]. The RS of R2 was lower than that of R1, whereas the RP showed the opposite result when the reactors operated with a similar ISC. One possible reason for these results was that the salt stress may cause cell decay, which would lead to a lower RS. As a result, R2 experienced a more severe inhibition because of the more rapid salt shock.

3.3. The enhanced adaptation strategies 3.3.1. Extra biomass addition (EBA) The ISC of R2 was maintained at 30 g L1 after day 155, with the effluent NH+4-N and NO 2 -N concentrations remaining at an alert level over which the anammox process will be inhibited [26]. The average NRENH and NRENO were 39% and 40%, respectively. In this situation, the test of EBA was conducted to investigate the impact of anammox biomass addition under high salinity. Every day, 10 mL of anammox sludge with high activity was added to the reactor after day 155. The NRR shifted to a higher level with the addition of fresh anammox sludge, with an SAA valued at approximately 18.63 mg N g1 VSS h1. As illustrated in Fig. 1, the NRR increased from 0.64 to 0.98 kg N m3 d1 by the addition of EBA. After the addition of the sludge for 8 days, the NRR was almost twice than that before the test. As a result, the NLR and NRR were 4.30 ± 0.54 and 1.49 ± 0.39 kg N m3 d1, respectively, whereas the effluent NH+4-N and NO 2 -N concentration decreased to 107.8 ± 32.5 and 89.9 ± 20.5 mg L1, respectively. This finding suggests that the addition of extra anammox biomass was effective at improving the anammox capacity under high salinity, and the mechanism may be due to two reasons: (1) Amplification of the dominant consortium and augmentation of its activity. Bartroll´ et al. [27] confirmed that bio-augmentation could enhance the ammonia oxidation bacteria activity in the nitrifier community. In the present study, the NRR of R2 was immediately enhanced after EBA. (2) Quorum-sensing effect [28].

3.3.2. Glycine betaine addition (GBA) A pre-experiment was conducted to determine the dosage of glycine betaine required to achieve optimal SAA. The SAA behavior was observed as the glycine betaine concentration was increased to 0.43 mmol L1; the maximum SAA (10.0% increase) was obtained when the glycine betaine concentration was 0.09 mmol L1 (data not shown). At the beginning of the test, the ISC was fixed at 10 g L1, along with the addition of 5 mmol L1 glycine betaine. The effluent NH+4-N and NO 2 -N concentrations reached 92.3 ± 52.4 and 15.1 ± 1.5 mg L1, respectively (Table 4). The influent NO 2 -N was nearly completely removed, but the influent NH+4-N was mainly retained. Moreover, the RS was 1.46 ± 0.87,

Fig. 2. The evolutions of the stoichiometric ratios in R1 (A) and R2 (B).

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and the RP was 0.10 ± 0.05, which significantly deviated from the stoichiometric ratios of 1.32 (RS) and 0.26 (RP). After day 6, the concentration of glycine betaine was decreased to 1 mmol L1. Then, the effluent NH+4-N and NO 2 -N concentrations decreased to 37.9 and 25.6 mg L1, respectively, on day 8. Unexpectedly, the effluent quality became worse under the same operating conditions, with a rapid rise in the substrate concentration. Very little NH+4-N was removed. Occasionally, the NH+4-N concentration in the effluent was higher than that in the influent, and 1 the maximum concentration of NO . Fur2 -N reached 110.3 mg L thermore, the values of RS and RP deviated more significantly from the aforementioned stoichiometric ratios. During the next period, from days 16–37, the concentration of glycine betaine was decreased to 0.1 mmol L1. The reactor did not show any signs of recovery during those days, and the effluent NH+4-N and NO 2 -N concentrations remained at high levels. Due to this situation, the ISC was adjusted to 5 g L1, and the reactor recovered slightly from the inhibition with RS and RP values of 1.07 ± 0.11 and 0.24 ± 0.05, respectively, which approached the theoretical stoichiometric ratios. The ISC was again increased to 10 g L1 from days 65 to 72 without any change in the glycine

betaine concentration, and the reactor capacity clearly improved. Then, the ISC was changed to 20 g L1, and the concentration of glycine betaine was decreased to 0.05 mmol L1. Then, the concentration of glycine betaine was slowly increased to 0.5 mmol L1 in the following days despite the high effluent level. The reactor did not reach a pseudo-steady state until day 145. By then, the ISC increased to 30 g L1, and the concentration of glycine betaine decreased to 0.1 mmol L1. As a result, the effluent NH+4-N and NO 2 -N concentrations reached their maximum values of 166.9 and 133.3 mg L1, respectively. Fig. 3 shows the performance of R3 during the GBA test. After comparing the NRR with R1 and R2 at the same ISC, an inferior performance of R3 was obtained. This finding may be attributed to the fact that glycine betaine is a type of organic substance that may inhibit the anammox microorganism, as discussed below. 3.3.3. Evaluation of the regulation measures The microorganisms could probably survive the high salinity in two ways. First, the microorganisms are able increase the concentration of ions such as K+ inside the cells to maintain a balance between the OP inside and outside the cell. Second, the

Table 4 Summary of anammox performance in the GBA experiment. Time (d)

Salinity (g L1)

Glycine betaine (mmol L1)

Eff. NH+4-N (mg L1)

Eff. NO 2 -N (mg L1)

RS

RP

1–5 6–15 16–37 38–64 65–72 73–81 82–89 90–103 104–121 122–145 146–163

10 10 10 5 10 20 20 20 20 20 30

5 1 0.1 0.1 0.1 0.05 0.1 0.15 0.2 0.5 0.1

92.3 ± 52.4 99.7 ± 79.8 159.2 ± 24.3 66.4 ± 29.4 34.8 ± 17.3 51.9 ± 32.2 87.4 ± 10.3 86.1 ± 19.9 145.3 ± 40.2 139.2 ± 18.6 153.1 ± 35.9

15.1 ± 1.5 58.2 ± 43.9 115.4 ± 30.3 58.7 ± 33.2 27.9 ± 12.9 40.7 ± 21.7 58.8 ± 7.7 59.9 ± 8.8 84.7 ± 10.2 95.1 ± 26.5 111.4 ± 17.3

1.46 ± 0.87 1.68 ± 1.44 2.48 ± 1.56 1.07 ± 0.11 1.28 ± 0.14 1.08 ± 0.16 1.03 ± 0.01 1.21 ± 0.32 1.26 ± 0.32 1.38 ± 0.81 1.12 ± 0.61

0.10 ± 0.05 0.11 ± 0.08 0.50 ± 0.31 0.24 ± 0.05 0.31 ± 0.06 0.19 ± 0.03 0.23 ± 0.02 0.31 ± 0.11 0.45 ± 0.20 0.45 ± 0.22 0.61 ± 0.25

Eff.: effluent. GBA: glycine betaine addition. + RP: the ratio of the production of NO 3 -N to the consumption of NH4-N. + RS: the conversion ratio of NO 2 -N/NH4-N.

Fig. 3. The anammox reactor performance in the GBA test.

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H. Chen et al. / Separation and Purification Technology 132 (2014) 584–592

Table 5 Summary of the research on the effect of salinity on anammox. No.

Salt

Reactor configuration

Salt concentration (g L1)

Effect

Reference

1

NaCl

Sequencing batch reactor (SBR)

5

Improving the retention of anammox biomass Specific anammox activity (SAA) slightly reduced Improving the retention of anammox biomass SAA gradually increased

[34]

10 2

NaCl

SBR

6

A stimulatory effect on the SAA was obtained (<6 g NaCl L1)

[35]

3

NaCl

Rotating biological contactor (RBC)

6 13.5 <20 20 30

Estimated IC50 The SAA values were nearly constant Decrease of the SAA Decrease of the SAA Non-adapted: 95% (SAA) Adapted: 59% (SAA)

[25]

4

NaCl

Vials (batch assays)

<8.78 13.46 >7.10 11.36 >7.45 14.9

The anammox activity was not affected 50% (IC50) Inhibitory effect 50% (IC50) Inhibitory effect 50% (IC50)

[36]

SBR

10, 30 45

Stable reactor performance was obtained The anammox activity was completely lost 5 days after the salt concentration increased to 45 g L1, but the inhibition was reversible The activity was lost The biomass was still active Inactivation

[3]

Na2SO4 KCl 5

90% NaCl and 10% KCl

60 75 90 6

NaCl

UASB

30

Non-adapted: 67.5% (SAA) Adapted: 45.1% (SAA)

[37]

7

NaCl

Fixed-bed anammox reactor (FBAR)

30 >30

Stable operation was obtained with a NRR of 1.7 kg N m3 d1 NRR sharply declined

[33]

8

NaCl

Up-flow column reactor (UFCR)

20 30

Significant inhibition A stable NRR of 4.5 ± 0.1 kg N m3 d1 was obtained

[38]

9

NaCl

SBR

8–10

A concentration of 10 g L1 NaCl did not have long-term negative effects on the activity or physical properties of the anammox sludge

[2]

10

NaCl

UASB

5–60

A concentration of 30 g L1 NaCl was the threshold value for the stability of the anammox process

[22]

11

NaCl

Sequencing batch biofilm reactor (SBBR)

10

The CANON process was stable at the concentration of 10 g NaCl L1

[31]

12

NaCl

UASB

10–30

Stable reactor performance was achieved at the concentration of 10 g NaCl L1 The NRR of R2 (rapid adaptation) was lower than that of R1 (gradual adaptation) A 32.5% increase of NRR was achieved by EBA at the concentration of 30 g NaCl L1 A maximum increase (10%) of SAA was achieved during the batch assays (30 g NaCl L1), but no benefit was obtained during the long-term test with GBA (5–30 g NaCl L1)

The present study

CANON: completely autotrophic nitrogen removal over nitrite. IC50: 50% inhibition concentration. NRR: nitrogen removal rate. SAA: specific anaerobic ammonium oxidation activity.

microorganisms accumulated compatible solutes might be another mechanism for maintaining osmotic balance. Glycine betaine is a compatible solute that can relieve salinity inhibition. However, the available research was focused on the system of methanogenesis [14], and the benefit of glycine betaine in a high-salinity anammox process remains a mystery. Although glycine betaine could be beneficial to coping with salinity inhibition, as an organic material in the reactor, GBA tended to cause intense competition for the nitrite substances between the denitrifiers and anammox bacteria, leading to the limitation of the growth of the anammox bacteria. The biodegradation of glycine betaine by anaerobic bacteria was observed by Thalasso et al. [29]. Despite the effectiveness of GBA in the batch assays, it was ineffective in the present study during the long-term operation. Three hypotheses may possibly explain this phenomenon: (1) glycine betaine could only promote the anammox pathway transiently, resembling the phenomenon explored in Vyrides et al. [14] (2) the dosage was inappropriate or (3) the feed mode should be adjusted. Therefore,

the operational optimization of GBA still requires further study regarding the dosage and feed mode. EBA clearly promoted the capacity of the reactor at a dosage of 10 mL of fresh sludge with high activity per day. However, further study is required to optimize the parameters, such as the dosage and frequency of the additives, in a long-term assay. 3.4. Summary of the anammox process under salinity conditions It is important to consider the impact of salinity as a potential inhibitory factor when employing the anammox process to treat salt-rich wastewater. Several researchers have explored anammox adaptation to high salinity (Table 5). Lay et al. [30] reported that the microorganism could adapt to the salinity when the salinity was less than 30 g L1. Zhang et al. [31] successfully started the completely autotrophic nitrogen removal over nitrite (CANON) process with a lab-scale sequencing batch biofilm reactor (SBBR) under a maximum salt concentration of 10 g NaCl L1 within

H. Chen et al. / Separation and Purification Technology 132 (2014) 584–592

118 days, with a maximum NRR of 0.072 kg TN m3 d1. Kartal et al. [32] operated an SBR for anammox treatment with a salt concentration of 30 g L1; the reported NRR was 1.0 kg N m3 d1. Windey et al. [25] attained an NRR of 0.609 kg N m3 d1, and Liu et al. [33] reached 1.7 kg N m3 d1 after exposing the reactor to 30 g NaCl L1. The average NRR in this study was 1.37 kg N m3 d1 at the concentration of 30 g NaCl L1, which falls within the ranges reported in the literature. There are three possible explanations for the NRR range previously reported. (1) The diversity of the anammox species: Dapena-Mora et al. [2] reported that Scalindua was halophilic, whereas Kuenenia was a freshwater species. (2) The type of sludge: flocculent, biofilm or biogranule forms were another possible reason as the water density was increased by increasing the salt concentration, and this variation could contribute to the washout of the biomass with a poor settling capacity, such as flocculent sludge. (3) The increase mode: gradual or rapid increases could affect the adaptation operation, as was demonstrated in the present study. The present study demonstrated that 15 g L1 NaCl did not show any negative impacts during the gradual adaptation test in R1. The NRR of the reactor under 15 g NaCl L1 was 3.48 ± 0.51 kg N m3 d1. Additionally, the NRR clearly decreased with the increase in ISC. Meanwhile, R2 operated steadily with an NRR of 3.71 ± 0.63 kg N m3 d1 under 10 g NaCl L1. The reactor performance deteriorated when the ISC increased to a higher level. It was proposed that low salinity was not detrimental to the anammox bacteria in this study but that the reactor performance would begin to degrade if the ISC was higher than 15 g L1. 4. Conclusions The present work proposed that the gradual adaptation reactor performed better than the rapid adaptation reactor in the selfadaptation strategy. In addition, the EBA-reactor was effective at resisting salinity stress in the enhanced adaptation strategy. For the GBA strategy, improvement of the SAA was attained during the batch assays, but ineffectiveness was observed during the long-term operation. Furthermore, the present work certified that the inhibition of salt on anammox activity was co-determined by ion species and osmotic pressure. Acknowledgements The authors wish to thank the National Natural Science Foundation of China (Nos. 51078121 and 51278162), the National Hightech Research and Development Program (863 Program) (No. 2011AA060801-6) and the project of Zhejiang Key Scientific and Technological Innovation Team (2010R50039) for partial support of this study. References [1] J.P. Bassin, R. Kleerebezem, G. Muyzer, A.S. Rosado, M.C.M. van Loosdrecht, M. Dezotti, Effect of different salt adaptation strategies on the microbial diversity, activity, and settling of nitrifying sludge in sequencing batch reactors, Appl. Microbiol. Biotechnol. 93 (2012) 1281–1294. [2] A. Dapena-Mora, J.L. Campos, A. Mosquera-Corral, R. Méndez, Anammox process for nitrogen removal from anaerobically digested fish canning effluents, Water Sci. Technol. 53 (2006) 265–274. [3] B. Kartal, M. Koleva, R. Arsov, W. van der Star, M.S.M. Jetten, M. Strous, Adaptation of a freshwater anammox population to high salinity wastewater, J. Biotechnol. 126 (2006) 546–553. [4] A.A. van de Graaf, P. de Bruijn, L.A. Robertson, M.S.M. Jetten, J. Gijs Kuenen, Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor, Microbiology 142 (1996) 2187–2196. [5] M. Strous, J.J. Heijnen, J.G. Kuenen, M.S.M. Jetten, The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammoniumoxidizing microorganisms, Appl. Microbiol. Biotechnol. 50 (1998) 589–596.

591

[6] B. Kartal, J.G. Kuenen, M.C.M. van Loosdrecht, Sewage treatment with anammox, Science 328 (2010) 702–703. [7] M.S.M. Jetten, I. Cirpus, B. Kartal, L. van Niftrik, K.T. van de Pas-Schoonen, O. Sliekers, S. Haaijer, W. van der Star, M. Schmid, J. van de Vossenberg, I. Schmidt, H. Harhangi, M.C.M. van Loosdrecht, J. Gijs Kuenen, H. Op den Camp, M. Strous, 1994–2004: 10 years of research on the anaerobic oxidation of ammonium, Biochem. Soc. Trans. 33 (2005) 119–123. [8] R.C. Jin, G.F. Yang, Q.Q. Zhang, C. Ma, J.J. Yu, B.S. Xing, The effect of sulfide inhibition on the ANAMMOX process, Water Res. 47 (2013) 1459–1469. [9] Q.Q. Zhang, G.F. Yang, H. Wang, K. Wu, R.C. Jin, P. Zheng, Estimating the recovery of ANAMMOX performance from inhibition by copper(II) and oxytetracycline (OTC), Sep. Purif. Technol. 113 (2013) 90–103. [10] C.J. Tang, P. Zheng, Q. Mahmood, The shear force amendments on the slugging behavior of upflow anammox granular sludge bed reactor, Sep. Purif. Technol. 69 (2009) 262–268. [11] R.C. Jin, G.F. Yang, J.J. Yu, P. Zheng, The inhibition of the anammox process: a review, Chem. Eng. J. 197 (2012) 67–79. [12] M. Soto, R. Mendéz, J.M. Lema, Methanogenic and nonmethanogenic activity tests: theoretical basis and experimental setup, Water Res. 27 (1993) 1361– 1376. [13] Y. Chen, J.J. Cheng, K.S. Creamer, Inhibition of anaerobic digestion process: a review, Bioresour. Technol. 99 (2008) 4044–4064. [14] I. Vyrides, H. Santos, A. Mingote, M.J. Ray, D.C. Stuckey, Are compatible solutes compatible with biological treatment of saline wastewater? Batch and continuous studies using submerged anaerobic membrane bioreactors (SAMBRs), Environ. Sci. Technol. 44 (2010) 7437–7442. [15] C.J. Tang, P. Zheng, T.T. Chen, J.Q. Zhang, Q. Mahmood, S. Ding, X.G. Chen, J.W. Chen, D.T. Wu, Enhanced nitrogen removal from pharmaceutical wastewater using SBA-ANAMMOX process, Water Res. 45 (2011) 201–210. [16] G.F. Yang, R.C. Jin, Reactivation of effluent granular sludge from a high-rate anammox reactor after storage, Biodegradation 24 (2013) 13–32. [17] G.F. Yang, W.M. Ni, K. Wu, H. Wang, B.E. Yang, X.Y. Jia, R.C. Jin, The effect of Cu(II) stress on the activity, performance and recovery on the anaerobic ammonium-oxidizing (anammox) process, Chem. Eng. J. 226 (2013) 39–45. [18] American Public Health Association (APHA), American Water Works Association (AWWA) and Water Environment Federation (AEF), Standard Methods for the Examination of Water and Wastewater, 21st ed. Washington, DC, USA, 2005. [19] P. Nelson, Biological Physics: Energy, Information, Life, WH Freeman & Co, New York, 2004. [20] A. Mosquera-Corral, F. González, J.L. Campos, R. Méndez, Partial nitrification in a SHARON reactor in the presence of salts and organic carbon compounds, Process Biochem. 40 (2005) 3109–3118. [21] J.M. Carvajal-Arroyo, W.J. Sun, R. Sierra-Alvarez, J.A. Field, Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents, Chemosphere 91 (2013) 22–27. [22] C. Ma, R.C. Jin, G.F. Yang, J.J. Yu, B.S. Xing, Q.Q. Zhang, Impacts of transient salinity shock loads on anammox process performance, Bioresour. Technol. 112 (2012) 124–130. [23] J.J. Yu, R.C. Jin, The ANAMMOX reactor under transient-state conditions: process stability with fluctuations of the nitrogen concentration, inflow rate, pH and sodium chloride addition, Bioresour. Technol. 119 (2012) 166–173. [24] G.F. Yang, X.L. Guo, S.X. Chen, J.H. Liu, L.X. Guo, R.C. Jin, The evolution of anammox performance and granular sludge characteristics under the stress of phenol, Bioresour. Technol. 132 (2013) 332–339. [25] K. Windey, I. De Bo, W. Verstraete, Oxygen-limited autotrophic nitrification– denitrification (OLAND) in a rotating biological contactor treating high-salinity wastewater, Water Res. 39 (2005) 4512–4520. [26] M. Strous, J.G. Kuenen, M.S.M. Jetten, Key physiology of anaerobic ammonium oxidation, Appl. Environ. Microbiol. 65 (1999) 3248–3250. [27] A. Bartroll´, J. Carrera, J. Pérez, Bioaugmentation as a tool for improving the start-up and stability of a pilot-scale partial nitrification bio-film airlift reactor, Bioresour. Technol. 102 (2011) 4370–4375. [28] R.C. Jin, Q.Q. Zhang, G.F. Yang, B.S. Xing, Y.X. Ji, H. Chen, Evaluating the recovery performance of the ANAMMOX process following inhibition by phenol and sulfide, Bioresour. Technol. 1421 (2013) 162–170. [29] F. Thalasso, J. van der Burgt, V.O. Flaherty, E. Colleran, Large scale anaerobic degradation of betaine, J. Chem. Technol. Biotechnol. 74 (1999) 1176–1182. [30] W.C.L. Lay, Y. Liu, A.G. Fane, Impacts of salinity on the performance of high retention membrane bioreactors for water reclamation: a review, Water Res. 44 (2010) 21–40. [31] Z.J. Zhang, S.H. Chen, P. Wu, L.F. Lin, H.Y. Luo, Start-up of the Canon process from activated sludge under salt stress in a sequencing batch biofilm reactor (SBBR), Bioresour. Technol. 101 (2010) 6309–6314. [32] B. Kartal, L. van Niftrik, O. Sliekers, M.C. Schmid, I. Schmidt, K. van de PasSchoonen, I. Cirpus, W. van der Star, M.C.M. van Loosdrecht, W. Abma, J. Gijs Kuenen, J.M. Mulder, M.S.M. Jetten, H. Op den Camp, M. Strous, J. van de Vossenberg, Application, eco-physiology and biodiversity of anaerobic ammonium-oxidizing bacteria, Rev. Environ. Sci. Biotechnol. 3 (2004) 255– 264. [33] C.L. Liu, T. Yamamoto, T. Nishiyama, T. Fujii, K. Furukawa, Effect of salt concentration in anammox treatment using non woven biomass carrier, J. Biosci. Bioeng. 107 (2009) 519–523.

592

H. Chen et al. / Separation and Purification Technology 132 (2014) 584–592

[34] I. Fernández, J.R. Vázquez-Padín, A. Mosquera-Corral, J.L. Campos, R. Méndez, Biofilm and granular systems to improve anammox biomass retention, Biochem. Eng. J. 42 (2008) 308–313. [35] A. Dapena-Mora, J.R. Vázquez-Padín, J.L. Campos, A. Mosquera-Corral, M.S.M. Jetten, R. Méndez, Monitoring the stability of an anammox reactor under high salinity conditions, Biochem. Eng. J. 51 (2010) 167–171. [36] A. Dapena-Mora, I. Fernandez, J.L. Campos, A. Mosquera-Corral, R. Méndez, M.S.M. Jetten, Evaluation of activity and inhibition effects on anammox

process by batch tests based on the nitrogen gas production, Enzyme Microb. Technol. 40 (2007) 859–865. [37] R.C. Jin, C. Ma, Q. Mahmood, G.F. Yang, P. Zheng, Anammox in a UASB reactor treating saline wastewater, Process Saf. Environ. Prot. 89 (2011) 342– 348. [38] J.C. Yang, L. Zhang, D. Hira, Y. Fukuzaki, K. Furukawa, Anammox treatment of high-salinity wastewater at ambient temperature, Bioresour. Technol. 102 (2011) 2367–2372.