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Insight into the short- and long-term effects of quinoline on anammox granules: Inhibition and acclimatization
Science of the Total Environment 651 (2019) 1294–1301
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Insight into the short- and long-term effects of quinoline on anammox granules: Inhibition and acclimatization Qian-Qian Chen 1, Lian-Zeng-Ji Xu 1, Zao-Zao Zhang, Fan-Qi Sun, Zhi-Jian Shi, Bao-Cheng Huang, Nian-Si Fan ⁎, Ren-Cun Jin ⁎ College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou 310036, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• The inhibitory effects of quinoline on anammox were investigated for the first time. • Gradient domestication was applied and toleration of 10 mg L−1 quinoline was achieved. • The content of EPS, heme c and diameter decreased after quinoline inhibition.
a r t i c l e
i n f o
Article history: Received 14 July 2018 Received in revised form 30 August 2018 Accepted 21 September 2018 Available online 22 September 2018 Editor: Zhen (Jason) He Keywords: Anammox Quinoline Inhibition Acute toxicity Granule characteristics
a b s t r a c t The short- and long-term influence of quinoline on the properties of anaerobic ammonium oxidation (anammox) biogranules was evaluated. During batch tests, the bioactivity of anammox granules in the presence of different quinoline concentrations was monitored, and the IC50 of quinoline was calculated to be 13.1 mg L−1 using a noncompetitive inhibition model. The response of anammox granules to pre-exposure to quinoline was dependent on metabolic status, and the presence of both quinoline and NO2−-N had a rapid detrimental effect, resulting in a 64.5% decrease within 12 h. During continuous-flow experiments, the nitrogen removal rate (NRR) of the reactor decreased sharply within 3 days in the presence of 10 mg L−1 quinoline and then was restored to 2.6 kg N m−3 d−1. In the presence of quinoline-induced stress, the specific anammox activity and levels of extracellular polymeric substance and heme c were decreased, while settling velocity persistently increased. After cultivation and acclimation obtained by adding a medium level of quinoline to the influent, the anammox granule sludge was able to tolerate 10 mg L−1 quinoline in 178 days. © 2018 Published by Elsevier B.V.
1. Introduction Coking wastewater is a type of industrial effluent originating from coal coking plants, and it contains a number of inorganic and organic pollutants, such as thiocyanide, phenols, naphthalene, ammonia, ⁎ Corresponding authors. E-mail addresses: [email protected] (N.-S. Fan), [email protected] (R.-C. Jin). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.scitotenv.2018.09.285 0048-9697/© 2018 Published by Elsevier B.V.
sulfide, cyanide, nitrogen heterocyclic compounds (NHCs), and polycyclic aromatic compounds (PHAs) (Kim et al., 2008; Lu et al., 2009). Quinoline is one type of NHC, and it has been observed at concentrations of up to 86.8 mg L−1 in coking wastewater (Wei et al., 2012). NHCs are refractory, poisonous and carcinogenic. Toh and Ashbolt (2002) believed that one of the most economic methods of ammonium removal was the combined partial nitritation and anaerobic ammonium oxidation (anammox) process by which ammonium was converted into nitrite by aerobic ammonium-oxidizing
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bacteria (AOB), and anammox bacteria combined the nitrite with residual ammonium to produce dinitrogen gas and nitrate (Fux and Siegrist, 2004). This novel process is superior to the conventional nitrification/ denitrification process used for wastewater due to a low aeration requirement, reduced sludge production, and no organic carbon consumption (Fux and Siegrist, 2004). Hence, the anammox process is a possible alternative for the removal of biological nitrogen from coking wastewater. Biological processes are susceptible to operational conditions (Jin et al., 2013). The aromatic compounds in coking wastewater would affect the metabolic processing of anammox biomass. Ramos et al. (2015) found that quinoline, at a concentration of 10 mg L−1, had a significant inhibitory effect during a short-term test. Yang et al. (2013) observed that the IC50 of phenol during a short-term test of the anammox process was 678.2 mg L−1. Phenol has a negative effect on metabolism during the anammox process and alters the bacterial community within the anammox reactor (Pereira et al., 2014). However, few studies have reported the effect of quinoline on the properties of anammox granules during long-term testing. Knowledge of inhibition of the anammox by quinoline is expected to expand the scope of application for the anammox process. Because of the large variation in the flow and composition of wastewaters, the anammox may be in the presence of famine conditions for days and, sometimes, weeks (Lu et al., 2007). Starvation can affect the performance of anammox. Furthermore, tests have demonstrated that anammox biomass is highly sensitive to nitrite toxicity during starvation (Wang et al., 2015). Quinoline can be degraded by specialized bacteria (Wang et al., 2001). Anammox bacteria might also be subject to quinoline exposure during a one-stage nitrification-anammox process or if there is no pretreatment. With regard to the characterization of the anammox system when under stress caused by exposure to quinoline, the goals of this study are to (1) examine the short-term impacts of quinoline on anammox bioactivity; (2) evaluate the long-term impacts on the performance of the anammox process and the properties of anammox granular sludge; and (3) demonstrate the feasibility of acclimatization to enhance the tolerance of anammox granules to quinoline. 2. Materials and methods 2.1. Synthetic wastewater The synthetic wastewater contained substrates, minerals, and trace elements, as described by Yang and Jin (2013). (NH4)2SO4 and NaNO2 served as sources of ammonium and nitrite at a molar ratio of 1:1. The pH of influent synthetic wastewater was kept at 7.72 ± 0.15 by the injection of 1 M HCl or NaOH. During continuous testing, the influent of the reactor was supplied with quinoline at a concentration that was set according to the test aims described below.
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respectively. The dominant anammox bacteria in the seeding sludge was derived from the genus Candidatus Kuenenia stuttgartiensis (Zhang et al., 2016).
2.3. Operational strategy The process of the entire experimental course was divided into four main periods (P0–P3) with regards to the concentration of quinoline in the influent, as described in Table 1. The influent concentrations of quinoline and nitrogen were adjusted based on the property of nitrogen in the anammox system. When the process performance began to strongly deteriorate, the NLR or quinoline concentration was decreased.
2.4. Batch test The batch tests were performed in serum bottles with a liquid phase volume of 120 mL in order to examine the short-term effects of quinoline on anammox activity and to evaluate the SAA of the anammox sludge. For the analysis of the short-term tests, anammox biomass was also collected from the parent reactor mentioned in 2.1. The initial pH was fixed at 7.4–7.6 by adding acid or hydroxide. The method of SAA determination used was as described by Yang and Jin (2013). The batch tests were conducted for 6 h in a thermostatic shaker at 35 ± 1 °C and 180 rpm. Three-milliliter test samples were then collected using an injection syringe and were stored at 4 °C for later determination of NH4+-N and NO2−-N bulk concentrations. The SAA was calculated using the equation SAA = MSCR/VSS, where MSCR is the maximum substrate consumption rate (Yang and Jin, 2013; Zhang et al., 2017b). The normalized anammox activity (NAA) was calculated according to the equation NAA = SAAinhibited/SAAcontrol × 100%, where SAAinhibited is the SAA of granules treated with quinoline, and SAAcontrol is the SAA of granules without quinoline treatment.
2.5. Analytical methods The concentrations of NH4+-N, NO2−-N, and NO3−-N of the water samples were routinely assessed spectrophotometrically by the phenol–hypochlorite method, the N-(1-naphthalene) –diaminoethane method, and the phenol disulphonic acid method, respectively (APHA et al., 2005). The heme c content was quantified using the methods described by Berry and Trumpower (1987). The “heating” method was utilized for the extraction of EPS (Sheng et al., 2006; Zhang et al., 2017a). The specific assay methods used to measure the carbohydrate and protein content in the soluble EPS (S-EPS) and bound EPS (B-EPS) fractions were as described by Wu et al. (2009). The total EPS was the sum of SEPS and B-EPS. The settling velocity of the granules was measured using the methods described by Shen et al. (2012).
2.2. Experimental configuration and seeding sludge The up-flow anaerobic sludge bed (UASB) reactor, R1, with a working volume of 1.0 L, was used to cultivate anammox granules. The UASB reactor was designed with an internal diameter of 70 mm. The reactor was covered with black cloth to avoid light-induced inhibition and then put in a thermostatic room at 35 ± 1 °C. The continuous testing reactor was inoculated with anammox granules that were harvested from a highly loaded laboratory-scale parent UASB reactor with a specific anammox activity (SAA) of approximately 176.7 ± 16.1 mg N g−1 VSS d−1. The suspended solid (SS) and volatile suspended solid (VSS) in the anammox granules in the reactors after inoculation were 30 and 22 g L−1, respectively. The diameter of an anammox granule was 2.62 ± 0.74 mm, and the settling velocity was 63.3 ± 15.9 m h−1. The content of the extracellular polymeric substance (EPS) and heme c were 432.2 ± 29.4 mg g−1 VSS and 3.91 ± 0.02 μmol g−1 VSS,
Table 1 Initial substrate and quinoline concentration during the batch test. Experimental
Short-term effects at fixed initial substrate level Short-term effects at various initial substrate level
Short-term effects at different exposure times
NH+ 4 -N (mg L−1)
NO− 2 -N (mg L−1)
Quinoline Exposure concentration time (mg L−1)
(h)
100
100
0,5,10,50,100
0
70 100 140 210 280 0 100 0
70 100 140 210 280 0 100 100
0 and 5
0 0 0 0 0 12,24,48,96
5
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2.6. Modified non-competitive inhibition model A modified non-competitive inhibition model (Eq. (1)) was used to describe the impact of quinoline inhibition on anammox: NAAð%Þ ¼ 100
1–
!
1
ð1Þ
b
1 þ ð½quinoline=aÞ
where NAA is the calculated inhibition response (%), [quinoline] is the quinoline concentration (mg L−1), a is the IC50 (mg L−1), and b is a fitting parameter. 3. Results and discussion 3.1. Short-term effects of quinoline on anammox activity 3.1.1. Acute toxicity tests The short-term impacts of quinoline-induced stress on anammox activity at an initial set substrate level were studied. As the quinoline dosage increased, the SAA noticeably decreased, with an initial substrate concentration of 200 mg L−1 TN (Fig. 1). According to the modified non-competitive inhibition model in Eq. (2), the IC50 was 13.07 mg L−1. The non-competitive inhibition model was generally used for evaluation of the relationship between the inhibitor concentration and SAA. Tang et al. (2018) demonstrated that Cu (II) uncompetitively inhibited on nitritation process. Ramos et al. (2015) researched the effects of o-cresol, p-nitrophenol, ochlorophenol, and quinoline on anammox, and the Loung, Aiba and noncompetitive models adjusted the inhibition of anammox by those aromatic compounds (Ramos et al., 2015). The IC50 was 31 ± 6 mg L−1 for quinoline (Ramos et al., 2015). The effects of phenol were investigated by Yang et al. (2013), and the IC50 was 678.2 mg L−1 (Yang et al., 2013). Those compounds that belong to the class of strongly toxic aromatic chemicals, such as phenol, o-cresol, pnitrophenol, o-chlorophenol and quinoline, had an inhibitory effect on anammox. NAAð%Þ ¼ 100
1–
!
1 1 þ ð½quinoline=13:07Þ
0:21
ðR2 ¼ 0:99Þ
ð2Þ
3.1.2. Short-term effects of quinoline on the kinetic characteristics of anammox The relationship between SAA and substrate concentration with or without quinoline is shown in Fig. 2. The SAA increased at first and then subsequently decreased as the substrate concentration increased. Without quinoline, the maximum SAA was 655.1 mg N g−1 VSS d−1 at a TN level of 200 mg L−1, and the minimum SAA reached 488.5 mg N g−1 VSS d−1 with 560 mg L−1 TN. Thus, the SAA was related to substrate concentration. The substrate affinity was less at a lower substrate concentration. Furthermore, the floc of granules contributed to lower substrate affinity. Wang et al. (2012) determined that the SAA was influenced by the morphology and diameter of granules. In the current study, the SAA at a quinoline level of 5 mg L−1 was lower than that of the control without quinoline, which also suggested that quinoline inhibited anammox. The SAA was 401.2 mg N g−1 VSS d−1 in anammox with concentrations of 560 mg L−1 TN and 5 mg L−1 quinoline; compared with the control without quinoline, the SAA was decreased by 17.9%. These results elucidated that the joint toxicity of quinoline and nitrite was synergistic. According to the report, anammox performance could be decreased by nitrite to a level exceeding the inhibitory critical value (Lotti et al., 2012). The inhibitory effect of NO2−-N on anammox has been widely studied (Dapena-Mora et al., 2007; Strous et al., 1999). When the NO2−-N concentration was 350 mg L−1, the activity of anammox decreased by
Fig. 1. Short-term effects of quinoline on anammox granules. A, relationship between the quinoline concentration and SAA; B, plot of the non-competitive inhibition model.
50% (Dapena-Mora et al., 2007). A concentration of 100 mg L−1 NO2−N markedly suppressed anammox performance (Strous et al., 1999). Moreover, the inhibition of anammox by nitrite is mediated by the concentration of free nitrous acid (FNA). Tang et al. (2010) found that 77.7 μg L−1 FNA led to a 12% decrease in SAA. In this study, the concentration of FNA was 65.7 μg L−1 under 560 mg L−1 TN at the beginning of the batch test, and the SAA of the biogranules might be inhibited by FNA. 3.1.3. Role of pre-exposure A series of experiments were designed to study the effect of preexposure conditions, as described in Table 1, on anammox. As is shown in Fig. 3, exposure times ranging from 12 to 96 h and a quinoline concentration of 5 mg L−1 were used for testing in the presence of substrate sufficient conditions, and the NAA used were 68.6 ± 1.7%, 57.1 ± 12.5%, 88.3 ± 3.7% and 49.4 ± 4.5%, respectively. The NAA decreased as the exposure time increased (apart from 48 h). Short exposure time had little impact in the presence of starvation conditions. The microbial effect of pollutants was related to the microbial metabolism. Compared with starvation conditions, there was a stronger inhibitory influence
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NO2−-N conversion to NH4+-N concentration (RS) of 1.11 ± 0.12 and of NO3−-N production to NH4+-N consumption (RP) of 0.18 ± 0.02 were both obtained. The RS and RP deviated from the theoretical values reported in other studies but were within normal ranges (Fernandes et al., 2018; Zhang et al., 2018).
− Fig. 2. Short-term effect of the initial NH+ 4 -N/NO2 -N concentration on the specific anammox activity (SAA) with/without quinoline.
due to exposure to Cu2+ in the presence of substrate (Chen et al., 2016). In this study, the effect of quinoline on anammox was not obvious due to the lower diffusion rate in granule sludge during shorter exposure times, and the suppression of SAA by quinoline was enhanced by starvation at longer exposure times. The SAA decreased sharply in the presence of quinoline and nitrite as exposure time increased. The NAA at different exposure times was 64.5 ± 1.5%, 52.6 ± 0.4%, 25.8 ± 6.9% and 6.2 ± 2.8%. Compared with the NAA in the presence of substrate sufficient conditions, the coexistence of NO2−-N and quinoline would aggravate the inhibition on anammox. 3.2. Long-term effects of quinoline on anammox performance 3.2.1. Performance of R1 without quinoline during the stabilizing phase (P0) During days 1–53 (P0), the influent of R1 was pumped without the addition of quinoline (Fig. 4). The R1 had a stable performance during this phase, and the nitrogen loading rate (NLR), nitrogen removal rate (NRR) and nitrogen removal efficiency (NRE) were 2.91 ± 0.24 kg m−3 d−1, 2.64 ± 0.10 kg m−3 d−1 and 91.24 ± 6.8%, respectively. As shown in Fig. 4, in the effluent, the NH4+-N and NO2−-N were 34.4 ± 19.4 mg L−1 and 12.4 ± 6.9 mg L−1, respectively, and with a shorter HRT, the reactor achieved a high removal capacity. Ratios of
Fig. 3. Effect of 5 mg L−1 quinoline on NAA in the presence of various levels of substrate.
3.2.2. Performance of R1 with high quinoline-induced stress (P1) During P1 (days 54–95), the anammox process was observed at different quinoline levels. Within three days in the presence of 1 mg L−1 quinoline, the NRE remained at 97.5 ± 2.4%. The performance of reactor was stable in this case. During days 57–75, the NRR and NRE of R1 were 2.8 ± 0.2 kg m−3 d−1 and 94.9 ± 2.3%, respectively, in the presence of 5 mg L−1 quinoline. During days 76–86, the NO2−-N of the effluent increased sharply from 27.2 to 71.4 mg L−1 in the presence of a quinoline concentration of 10 mg L−1, and the NRR continued to decrease until it reached 2.1 kg m−3 d−1. The RS and RP were 0.95 ± 0.11 and 0.15 ± 0.02, respectively, on days 57–75, and the abnormal reaction ratio indicated that the metabolic process of anammox was disturbed by the presence of quinoline. To recover the performance of reactor, the quinoline concentration in the influent was decreased to 5 mg L−1. However, the performance of the nitrogen removal process continued to deteriorate. The NH4+-N and NO2−-N concentrations in the effluent increased to 102.5 and 138.8 mg L−1, respectively, and the minimal NRR was 1.6 kg m−3 d−1. This result showed that anammox performance could not be restored, even if subjected to only a short period of quinoline-induced stress. The degree of disorder of RS decreased to 0.87 ± 0.12, and the RP was maintained at 0.15 ± 0.02. It is reasonable to assume that the accumulation of quinoline caused metabolic disorder. 3.2.3. Performance of R1 during the recovery phase (P2) On day 96, to accelerate the restoration of anammox performance, the NH4+-N/NO2−-N and quinoline concentrations in the influent were decreased to 210 and 0 mg L−1, respectively. The performance of R1 was gradually restored, and the effluent NO2−-N concentration was decreased to 18 mg L−1. Subsequently, the influent NH4+-N and NO2−-N concentrations were increased to 280 mg L−1, and the function of reactor stabilized on day 116. The average effluent NO2−-N and NRE were 33.3 ± 12.5 mg L−1 and 84.0 ± 4.9%, respectively, during days 116–129. The performance of R1 still had not fully recovered compared to that of P0 and P1, even though the NO2−-N concentration in the effluent was far below 70 mg L−1. After discontinuation of quinoline addition, RS and RP recovered to 1.02 ± 0.11 and 0.16 ± 0.04, respectively. The performance of the nitrogen removal process and the reaction ratio also recovered simultaneously after long-term acclimatization during this phase. 3.2.4. Performance of R1 after acclimatization with various levels of quinoline (P3) The resistance of anammox bacteria to quinoline was enhanced via sludge acclimatization. As shown in Table 2, the influent quinoline concentration was gradually increased from 1 to 5 mg L−1, and the NRR and NRE of R1 were 2.7 ± 0.2 kg m−3 d−1 and 92.1 ± 4.3%, respectively. Although the influent contained quinoline, the performance of the reactor was gradually restored. The NRR was stabilized in the presence of 10 mg L−1 quinoline within 12 days. After long-term acclimatization, anammox constituents had certain ability to tolerate quinoline. The degrees of disorder of RS and RP (1.05 ± 0.15 and 0.23 ± 0.09), also decreased. Taking what is above-mentioned into account, it could provide new insights into the operation of anammox processes for treating wastewater containing quinoline. Pre-treatment is necessary to control quinoline concentration in a proper range. And gradual acclimatization is also a crucial step to treat coking wastewater according to our longterm effects experiments. What's more, water quality monitoring and early warning need to be intensified when necessary.
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− − Fig. 4. Performance of the anammox reactor. A, the concentration of NH+ 4 -N, NO2 -N and NO3 -N in the influent and effluent; B, the NLR, NRR and NRE of the anammox reactor in the presence of different concentrations of quinoline.
3.3. Long-term effects of quinoline on the properties of anammox granules 3.3.1. SAA The SAA and biomass co-determined the anammox performance (Tsushima et al., 2007). As is depicted in Fig. 5, the SAA had a various
response under different quinoline concentration. Critical points were selected at which to evaluate the SAA in the continuous flow reactor. The SAA of inoculated sludge was 176.7 ± 16.1 mg N g−1 VSS d−1. On day 77, the SAA decreased to 127.6 ± 4.5 mg N g−1 VSS d−1 in the presence of 5 mg L−1 quinoline, and the percent of loss was 27.8%. This result
Table 2 Testing conditions and performance of the anammox reactor during different operational phases. Phase
Experimental aim
Operation time
− NH+ 4 -N/NO2 -N concentration (mg L−1)
Quinoline concentration (mg L−1)
NRE: (%)
NLR: (kg m−3 d−1)
NRR: (kg m−3 d−1)
P0 P1
Performance without quinoline Performance with increasing quinoline quickly
P2 P3
Performance recovery without quinoline Performance with increasing quinoline slowly
1–53 d 54–56 d 57–75 d 76–84 d 85–95 d 96–138 d 139–152 d 153–166 d 167–180 d 181–192 d 193–204 d 205–219 d 220–232 d
280 280 280 280 280 140,210,280 280 280 280 280 280 280 280
0 1 5 10 5 0 1 2 3 4 5 8 10
92.2 ± 3.8 97.5 ± 2.4 94.9 ± 2.3 76.1 ± 10.5 63.5 ± 6.4 80.1 ± 10.0 85.0 ± 10.4 91.0 ± 3.5 91.4 ± 4.3 94.2 ± 1.5 97.1 ± 2.7 89.8 ± 6.1 92.2 ± 5.3
2.8 ± 0.2 2.4 ± 0.0 2.9 ± 0.2 2.8 ± 0.2 2.8 ± 0.2 2.4 ± 0.5 2.8 ± 0.2 2.8 ± 0.2 3.0 ± 0.2 2.8 ± 0.1 2.6 ± 0.5 2.5 ± 0.6 2.6 ± 0.3
2.6 ± 0.2 2.3 ± 0.0 2.8 ± 0.2 2.1 ± 0.4 1.8 ± 0.2 1.9 ± 0.5 2.3 ± 0.3 2.5 ± 0.3 2.8 ± 0.2 2.7 ± 0.1 2.6 ± 0.4 2.3 ± 0.6 2.4 ± 0.3
NRE: nitrogen removal efficiency; NLR: nitrogen loading rate; NRR: nitrogen removal rate.
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demonstrated that anammox was suppressed by a low concentration of quinoline. As the reactor was in a state of functional redundancy, the NRR or NLR had changed very little. Therefore, the removal of nitrogen was not obviously affected when accompanied with a decrease in SAA. There was a persistent effect of quinolone on the activity of the destroyed anammox granules. Thus, anammox performance and bioactivity did not achieve an immediate recovery after removal of quinoline from the influent. The minimal SAA was 29.7 ± 7.5 mg N g−1 VSS d−1 without quinoline in P2 (day 111). In P3 (days 139–232), a strategy of gradually increasing the concentration was used to cultivate and acclimate the granules to low concentrations of quinoline. The performance and SAA were still gradually increased, though 1 mg L−1 quinoline was added again on day 129. The SAA recovered to 97.3% of the initial level after long-term operation of the reactor (day 220). The influent quinoline concentration was increased to 10 mg L−1, and the reactor demonstrated stable performance. Therefore, the reactor was able to tolerate 10 mg L−1 quinoline after a long-term acclimatization. 3.3.2. EPS and settling velocity EPS plays a crucial role in the resistance of toxic substances. EPS was secreted at a higher level by the microbial cells in activated sludge, biogranules and biofilms in order to provide for selfdefense from toxic substances such as heavy metals (Li and Yu, 2014). Zhang et al. (2015) regarded EPS as a distinct self-response to Cu(II) stress. The level of EPS (PN and PS) is shown in Fig. 4.6A, and the trend of its production during the entire test was first of decrease and then increase. The EPS content decreased by 49.3% compared to that obtained without quinoline accretion on day 152. Later, the increased concentration of quinoline stimulated the production of EPS, so the EPS content gradually increased after day 152. At the end of the experiment, the level of EPS was back to 60.4% of the initial value. The changing trend of EPS production in this study was inconsistent with our previous study, where it showed an increase when anammox sludge was exposed to SCN− (Chen et al., 2017a). Xu et al. (2018) found the EPS content varied obviously under different shock tests. There are many factors may affect the content of EPS, such as the microbial community structure, temperature, substrate (Nichols et al., 2004). Therefore, the trend of EPS content in this study needs a further exploration. A suitable settling velocity has been associated with sludge retention. In this study, the initial settling velocity was 63.9 ± 15.9 m h−1, and the value had increased to 71.3 ± 8.2 m h−1 by day 129. The PN/ PS of EPS was in line with the settling velocity and intensity (Franco et al., 2006; Tang et al., 2011; Wu et al., 2009). Franco et al. (2006) found that anammox granule sludge possessed a better settleability
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when it had a relatively low PN/PS ratio. On day 167, the minimum PN/PS ratio was 7.1, with a maximum settleability of 72.1 ± 12.1 m h−1. Interestingly, an opposing trend was observed in that the settling velocity increased with the PN/PS ratio, increasing after day 129. The maximum of PN/PS ratio was 19.5 on day 129, with a settleability of 70.7 ± 11.5 m h−1. The degree of the relationship between PN/PS and sludge settleability warrants further investigation. 3.3.3. Heme c Heme is an essential part of the pivotal anammox enzymes, including hydrazine synthesis (HZS), hydroxylamine oxidoreductase (HAO), and hydrazine oxidase (HZO) (Jetten et al., 2009). Anammox bacteria are rich in heme, which is a vital cofactor of cytochrome enzymes as it is involved in energy metabolism. Heme c is hypothesized to play an essential role in the carmine color of the anammox sludge, and the content of heme c might be related to bioactivity (Franco et al., 2006). During the course of the entire experiment, the trend of the change of the heme c level was consistent with that of the SAA. The heme c level had fallen only slightly from the initial value due to the addition of quinoline, from 3.9 ± 0.02 to 3.8 ± 0.04 μmol g−1 VSS, which had the synchronous trend with SAA by day 81. And the loss of SAA was 54.2% at that time. This result indicated that the anammox activity was enormously impacted by the presence of quinoline inhibition. The level of heme c had decreased to 1.3 ± 0.02 μmol g−1 VSS reaching the minimum value on day 129. Furthermore, SAA recovered to some extent from that moment on. On day 208, the heme c level had recovered to 3.1 ± 0.5 μmol g−1 VSS. Heme c content is related to NRR and SAA (Tang et al., 2011). Xu et al. (2019) demonstrated that the variation trend of heme c content in the anammox system was synchronous with NRR and SAA under NiO NPs exposure. In this study, the recovery of heme c content was slower than that of the SAA. More recently, a similar phenomenon was observed in a SCN− shock test, and the restoration time of heme c was considerably longer than that of SAA (Chen et al., 2017b). 3.3.4. Granule diameter The performance of the anammox granules is an essential factor affecting the reactor process. The granule diameter is dependent upon many factors, such as the reactor configuration, substrate loading rate, solid retention time, HRT, temperature, and pH (Marshall et al., 1984). The specific surface area of particle isn't too high to ensure the resistance to the load and hydraulic shocks (Yang and Jin, 2013). In this study, the sludge diameters determined under various quinoline concentrations are shown in Fig. 6D. In the whole anammox operation, the diameters of the anammox granules varied from 2.6 ± 0.7 to 2.3 ± 0.6 mm, with an average value of 2.4 ± 0.7 mm. In the initial 152 days, the granules diameter decreased from 2.6 ± 0.7 to 2.0 ± 0.52 mm due to the addition of quinoline. This result indicated that the quinoline presented in the reactor may partially destroy the granule structure. After 152 days, the diameter was 2.24 ± 0.6 mm and was hardly changed in the presence of quinoline. On day 208, the diameter was 76.9% of the initial value, and nearly 95% of the anammox granules had diameters of less than 2.5 mm. 4. Conclusion
Fig. 5. The SAA of anammox granules in different phases.
The short-term effects of quinoline on anammox were assessed based on quinoline concentration, substrate level, pre-exposure time and the presence of starvation conditions. The inhibition of anammox by quinoline was non-competitive, and the IC50 of quinoline on anammox mixed culture was 13.07 mg L−1. The anammox consortia could tolerate 10 mg L−1 quinoline by means of the gradient acclimatization during the continuous assays. The levels of EPS and heme c and granule diameter decreased by 49.3%, 67.1% and 23.2%, respectively, after inhibition with quinoline. These results may have a guiding role
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Fig. 6. The characteristics of anammox granules at different phases. A, EPS amounts and compositions; B, Heme c levels; C, Settling velocity; D, Diameter.
in propelling the application of the anammox process to treat wastewater containing quinoline.
Acknowledgements The authors wish to thank the Natural Science Foundation of China (No. 51578204) for the partial support of this study. References APHA, AWWA, AEF, 2005. Standard Methods for the Examination of Water and Wastewater. twenty first ed. American Public Health Association, Washington, DC, USA. Berry, E.A., Trumpower, B.L., 1987. Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem. 161, 1–15. Chen, H., Chen, Q.Q., Jiang, X.Y., Hu, H.Y., Shi, M.L., Jin, R.C., 2016. Insight into the shortand long-term effects of Cu(II) on denitrifying biogranules. J. Hazard. Mater. 304, 448–456. Chen, Q.Q., Chen, H., Zhang, Z.Z., Guo, L.X., Jin, R.C., 2017a. Effects of thiocyanate on granule-based anammox process and implications for regulation. J. Hazard. Mater. 321, 81–91. Chen, Q.Q., Sun, F.Q., Guo, Q., Shen, Y.Y., Zhu, W.Q., Jin, R.C., 2017b. Process stability in an anammox UASB reactor with individual and combined thiocyanate and hydraulic shocks. Sep. Purif. Technol. 173, 165–173. Dapena-Mora, A., Fernandez, I., Campos, J.L., Mosquera-Corral, A., Mendez, R., Jetten, M.S.M., 2007. Evaluation of activity and inhibition effects on anammox process by batch tests based on the nitrogen gas production. Enzym. Microb. Technol. 40, 859–865. Fernandes, L.D., Pereira, A.D., Leal, C.D., Davenport, R., Werner, D., Mota, C.R., BressaniRibeiro, T., Chernicharo, C.A.D., de Araujo, J.C., 2018. Effect of temperature on microbial diversity and nitrogen removal performance of an anammox reactor treating anaerobically pretreated municipal wastewater. Bioresour. Technol. 258, 208–219. Franco, A., Roca, E., Lema, J.M., 2006. Granulation in high-load denitrifying upflow sludge bed (USB) pulsed reactors. Water Res. 40, 871–880.
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Q.-Q. Chen et al. / Science of the Total Environment 651 (2019) 1294–1301 Tang, C.J., Zheng, P., Hu, B.L., Chen, J.W., Wang, C.H., 2010. Influence of substrates on nitrogen removal performance and microbiology of anaerobic ammonium oxidation by operating two UASB reactors fed with different substrate levels. J. Hazard. Mater. 181, 19–26. Tang, C.J., Zheng, P., Wang, C.H., Mahmood, Q., Zhang, J.Q., Chen, X.G., Zhang, L., Chen, J.W., 2011. Performance of high-loaded ANAMMOX UASB reactors containing granular sludge. Water Res. 45, 135–144. Tang, C.J., Duan, C.S., Liu, P., Chai, X.L., Min, X.B., Wang, S., Xiao, R.Y., Wei, Z.S., 2018. Inhibition kinetics of ammonium oxidizing bacteria under Cu(II) and As(III) stresses during the nitritation process. Chem. Eng. J. 352, 811–817. Toh, S.K., Ashbolt, N.J., 2002. Adaptation of anaerobic ammonium-oxidising consortium to synthetic coke-ovens wastewater. Appl. Microbiol. Biotechnol. 59, 344–352. Tsushima, I., Ogasawara, Y., Kindaichi, T., Satoh, H., Okabe, S., 2007. Development of highrate anaerobic ammoniumoxidizing (anammox) biofilm reactors. Water Res. 41, 1623–1634. Wang, J.L., Han, L.P., Shi, H.C., Qian, Y., 2001. Biodegradation of quinoline by gel immobilized Burkholderia sp. Chemosphere 44, 1041–1046. Wang, L., Zheng, P., Chen, T.T., Chen, J.W., Xing, Y.J., Ji, Q.X., Zhang, M., Zhang, J.Q., 2012. Performance of autotrophic nitrogen removal in the granular sludge bed reactor. Bioresour. Technol. 123, 78–85. Wang, Y.Y., Zhou, S., Wang, H., Ye, L., Qin, J., Lin, X.M., 2015. Comparison of endogenous metabolism during long-term anaerobic starvation of nitrite/nitrate cultivated denitrifying phosphorus removal sludges. Water Res. 68, 374–386. Wei, X.X., Zhang, Z.Y., Fan, Q.L., Yuan, X.Y., Guo, D.S., 2012. The effect of treatment stages on the coking wastewater hazardous compounds and their toxicity. J. Hazard. Mater. 239, 135–141. Wu, J., Zhou, H., Li, H., Zhang, P., Jiang, J., 2009. Impacts of hydrodynamic shear force on nucleation of flocculent sludge in anaerobic reactor. Water Res. 43, 3029–3036.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Insight into the short- and long-term effects of quinoline on anammox granules: Inhibition and acclimatization Qian-Qian Chen 1, Lian-Zeng-Ji Xu 1, Zao-Zao Zhang, Fan-Qi Sun, Zhi-Jian Shi, Bao-Cheng Huang, Nian-Si Fan ⁎, Ren-Cun Jin ⁎ College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou 310036, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• The inhibitory effects of quinoline on anammox were investigated for the first time. • Gradient domestication was applied and toleration of 10 mg L−1 quinoline was achieved. • The content of EPS, heme c and diameter decreased after quinoline inhibition.
a r t i c l e
i n f o
Article history: Received 14 July 2018 Received in revised form 30 August 2018 Accepted 21 September 2018 Available online 22 September 2018 Editor: Zhen (Jason) He Keywords: Anammox Quinoline Inhibition Acute toxicity Granule characteristics
a b s t r a c t The short- and long-term influence of quinoline on the properties of anaerobic ammonium oxidation (anammox) biogranules was evaluated. During batch tests, the bioactivity of anammox granules in the presence of different quinoline concentrations was monitored, and the IC50 of quinoline was calculated to be 13.1 mg L−1 using a noncompetitive inhibition model. The response of anammox granules to pre-exposure to quinoline was dependent on metabolic status, and the presence of both quinoline and NO2−-N had a rapid detrimental effect, resulting in a 64.5% decrease within 12 h. During continuous-flow experiments, the nitrogen removal rate (NRR) of the reactor decreased sharply within 3 days in the presence of 10 mg L−1 quinoline and then was restored to 2.6 kg N m−3 d−1. In the presence of quinoline-induced stress, the specific anammox activity and levels of extracellular polymeric substance and heme c were decreased, while settling velocity persistently increased. After cultivation and acclimation obtained by adding a medium level of quinoline to the influent, the anammox granule sludge was able to tolerate 10 mg L−1 quinoline in 178 days. © 2018 Published by Elsevier B.V.
1. Introduction Coking wastewater is a type of industrial effluent originating from coal coking plants, and it contains a number of inorganic and organic pollutants, such as thiocyanide, phenols, naphthalene, ammonia, ⁎ Corresponding authors. E-mail addresses: [email protected] (N.-S. Fan), [email protected] (R.-C. Jin). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.scitotenv.2018.09.285 0048-9697/© 2018 Published by Elsevier B.V.
sulfide, cyanide, nitrogen heterocyclic compounds (NHCs), and polycyclic aromatic compounds (PHAs) (Kim et al., 2008; Lu et al., 2009). Quinoline is one type of NHC, and it has been observed at concentrations of up to 86.8 mg L−1 in coking wastewater (Wei et al., 2012). NHCs are refractory, poisonous and carcinogenic. Toh and Ashbolt (2002) believed that one of the most economic methods of ammonium removal was the combined partial nitritation and anaerobic ammonium oxidation (anammox) process by which ammonium was converted into nitrite by aerobic ammonium-oxidizing
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bacteria (AOB), and anammox bacteria combined the nitrite with residual ammonium to produce dinitrogen gas and nitrate (Fux and Siegrist, 2004). This novel process is superior to the conventional nitrification/ denitrification process used for wastewater due to a low aeration requirement, reduced sludge production, and no organic carbon consumption (Fux and Siegrist, 2004). Hence, the anammox process is a possible alternative for the removal of biological nitrogen from coking wastewater. Biological processes are susceptible to operational conditions (Jin et al., 2013). The aromatic compounds in coking wastewater would affect the metabolic processing of anammox biomass. Ramos et al. (2015) found that quinoline, at a concentration of 10 mg L−1, had a significant inhibitory effect during a short-term test. Yang et al. (2013) observed that the IC50 of phenol during a short-term test of the anammox process was 678.2 mg L−1. Phenol has a negative effect on metabolism during the anammox process and alters the bacterial community within the anammox reactor (Pereira et al., 2014). However, few studies have reported the effect of quinoline on the properties of anammox granules during long-term testing. Knowledge of inhibition of the anammox by quinoline is expected to expand the scope of application for the anammox process. Because of the large variation in the flow and composition of wastewaters, the anammox may be in the presence of famine conditions for days and, sometimes, weeks (Lu et al., 2007). Starvation can affect the performance of anammox. Furthermore, tests have demonstrated that anammox biomass is highly sensitive to nitrite toxicity during starvation (Wang et al., 2015). Quinoline can be degraded by specialized bacteria (Wang et al., 2001). Anammox bacteria might also be subject to quinoline exposure during a one-stage nitrification-anammox process or if there is no pretreatment. With regard to the characterization of the anammox system when under stress caused by exposure to quinoline, the goals of this study are to (1) examine the short-term impacts of quinoline on anammox bioactivity; (2) evaluate the long-term impacts on the performance of the anammox process and the properties of anammox granular sludge; and (3) demonstrate the feasibility of acclimatization to enhance the tolerance of anammox granules to quinoline. 2. Materials and methods 2.1. Synthetic wastewater The synthetic wastewater contained substrates, minerals, and trace elements, as described by Yang and Jin (2013). (NH4)2SO4 and NaNO2 served as sources of ammonium and nitrite at a molar ratio of 1:1. The pH of influent synthetic wastewater was kept at 7.72 ± 0.15 by the injection of 1 M HCl or NaOH. During continuous testing, the influent of the reactor was supplied with quinoline at a concentration that was set according to the test aims described below.
1295
respectively. The dominant anammox bacteria in the seeding sludge was derived from the genus Candidatus Kuenenia stuttgartiensis (Zhang et al., 2016).
2.3. Operational strategy The process of the entire experimental course was divided into four main periods (P0–P3) with regards to the concentration of quinoline in the influent, as described in Table 1. The influent concentrations of quinoline and nitrogen were adjusted based on the property of nitrogen in the anammox system. When the process performance began to strongly deteriorate, the NLR or quinoline concentration was decreased.
2.4. Batch test The batch tests were performed in serum bottles with a liquid phase volume of 120 mL in order to examine the short-term effects of quinoline on anammox activity and to evaluate the SAA of the anammox sludge. For the analysis of the short-term tests, anammox biomass was also collected from the parent reactor mentioned in 2.1. The initial pH was fixed at 7.4–7.6 by adding acid or hydroxide. The method of SAA determination used was as described by Yang and Jin (2013). The batch tests were conducted for 6 h in a thermostatic shaker at 35 ± 1 °C and 180 rpm. Three-milliliter test samples were then collected using an injection syringe and were stored at 4 °C for later determination of NH4+-N and NO2−-N bulk concentrations. The SAA was calculated using the equation SAA = MSCR/VSS, where MSCR is the maximum substrate consumption rate (Yang and Jin, 2013; Zhang et al., 2017b). The normalized anammox activity (NAA) was calculated according to the equation NAA = SAAinhibited/SAAcontrol × 100%, where SAAinhibited is the SAA of granules treated with quinoline, and SAAcontrol is the SAA of granules without quinoline treatment.
2.5. Analytical methods The concentrations of NH4+-N, NO2−-N, and NO3−-N of the water samples were routinely assessed spectrophotometrically by the phenol–hypochlorite method, the N-(1-naphthalene) –diaminoethane method, and the phenol disulphonic acid method, respectively (APHA et al., 2005). The heme c content was quantified using the methods described by Berry and Trumpower (1987). The “heating” method was utilized for the extraction of EPS (Sheng et al., 2006; Zhang et al., 2017a). The specific assay methods used to measure the carbohydrate and protein content in the soluble EPS (S-EPS) and bound EPS (B-EPS) fractions were as described by Wu et al. (2009). The total EPS was the sum of SEPS and B-EPS. The settling velocity of the granules was measured using the methods described by Shen et al. (2012).
2.2. Experimental configuration and seeding sludge The up-flow anaerobic sludge bed (UASB) reactor, R1, with a working volume of 1.0 L, was used to cultivate anammox granules. The UASB reactor was designed with an internal diameter of 70 mm. The reactor was covered with black cloth to avoid light-induced inhibition and then put in a thermostatic room at 35 ± 1 °C. The continuous testing reactor was inoculated with anammox granules that were harvested from a highly loaded laboratory-scale parent UASB reactor with a specific anammox activity (SAA) of approximately 176.7 ± 16.1 mg N g−1 VSS d−1. The suspended solid (SS) and volatile suspended solid (VSS) in the anammox granules in the reactors after inoculation were 30 and 22 g L−1, respectively. The diameter of an anammox granule was 2.62 ± 0.74 mm, and the settling velocity was 63.3 ± 15.9 m h−1. The content of the extracellular polymeric substance (EPS) and heme c were 432.2 ± 29.4 mg g−1 VSS and 3.91 ± 0.02 μmol g−1 VSS,
Table 1 Initial substrate and quinoline concentration during the batch test. Experimental
Short-term effects at fixed initial substrate level Short-term effects at various initial substrate level
Short-term effects at different exposure times
NH+ 4 -N (mg L−1)
NO− 2 -N (mg L−1)
Quinoline Exposure concentration time (mg L−1)
(h)
100
100
0,5,10,50,100
0
70 100 140 210 280 0 100 0
70 100 140 210 280 0 100 100
0 and 5
0 0 0 0 0 12,24,48,96
5
1296
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2.6. Modified non-competitive inhibition model A modified non-competitive inhibition model (Eq. (1)) was used to describe the impact of quinoline inhibition on anammox: NAAð%Þ ¼ 100
1–
!
1
ð1Þ
b
1 þ ð½quinoline=aÞ
where NAA is the calculated inhibition response (%), [quinoline] is the quinoline concentration (mg L−1), a is the IC50 (mg L−1), and b is a fitting parameter. 3. Results and discussion 3.1. Short-term effects of quinoline on anammox activity 3.1.1. Acute toxicity tests The short-term impacts of quinoline-induced stress on anammox activity at an initial set substrate level were studied. As the quinoline dosage increased, the SAA noticeably decreased, with an initial substrate concentration of 200 mg L−1 TN (Fig. 1). According to the modified non-competitive inhibition model in Eq. (2), the IC50 was 13.07 mg L−1. The non-competitive inhibition model was generally used for evaluation of the relationship between the inhibitor concentration and SAA. Tang et al. (2018) demonstrated that Cu (II) uncompetitively inhibited on nitritation process. Ramos et al. (2015) researched the effects of o-cresol, p-nitrophenol, ochlorophenol, and quinoline on anammox, and the Loung, Aiba and noncompetitive models adjusted the inhibition of anammox by those aromatic compounds (Ramos et al., 2015). The IC50 was 31 ± 6 mg L−1 for quinoline (Ramos et al., 2015). The effects of phenol were investigated by Yang et al. (2013), and the IC50 was 678.2 mg L−1 (Yang et al., 2013). Those compounds that belong to the class of strongly toxic aromatic chemicals, such as phenol, o-cresol, pnitrophenol, o-chlorophenol and quinoline, had an inhibitory effect on anammox. NAAð%Þ ¼ 100
1–
!
1 1 þ ð½quinoline=13:07Þ
0:21
ðR2 ¼ 0:99Þ
ð2Þ
3.1.2. Short-term effects of quinoline on the kinetic characteristics of anammox The relationship between SAA and substrate concentration with or without quinoline is shown in Fig. 2. The SAA increased at first and then subsequently decreased as the substrate concentration increased. Without quinoline, the maximum SAA was 655.1 mg N g−1 VSS d−1 at a TN level of 200 mg L−1, and the minimum SAA reached 488.5 mg N g−1 VSS d−1 with 560 mg L−1 TN. Thus, the SAA was related to substrate concentration. The substrate affinity was less at a lower substrate concentration. Furthermore, the floc of granules contributed to lower substrate affinity. Wang et al. (2012) determined that the SAA was influenced by the morphology and diameter of granules. In the current study, the SAA at a quinoline level of 5 mg L−1 was lower than that of the control without quinoline, which also suggested that quinoline inhibited anammox. The SAA was 401.2 mg N g−1 VSS d−1 in anammox with concentrations of 560 mg L−1 TN and 5 mg L−1 quinoline; compared with the control without quinoline, the SAA was decreased by 17.9%. These results elucidated that the joint toxicity of quinoline and nitrite was synergistic. According to the report, anammox performance could be decreased by nitrite to a level exceeding the inhibitory critical value (Lotti et al., 2012). The inhibitory effect of NO2−-N on anammox has been widely studied (Dapena-Mora et al., 2007; Strous et al., 1999). When the NO2−-N concentration was 350 mg L−1, the activity of anammox decreased by
Fig. 1. Short-term effects of quinoline on anammox granules. A, relationship between the quinoline concentration and SAA; B, plot of the non-competitive inhibition model.
50% (Dapena-Mora et al., 2007). A concentration of 100 mg L−1 NO2−N markedly suppressed anammox performance (Strous et al., 1999). Moreover, the inhibition of anammox by nitrite is mediated by the concentration of free nitrous acid (FNA). Tang et al. (2010) found that 77.7 μg L−1 FNA led to a 12% decrease in SAA. In this study, the concentration of FNA was 65.7 μg L−1 under 560 mg L−1 TN at the beginning of the batch test, and the SAA of the biogranules might be inhibited by FNA. 3.1.3. Role of pre-exposure A series of experiments were designed to study the effect of preexposure conditions, as described in Table 1, on anammox. As is shown in Fig. 3, exposure times ranging from 12 to 96 h and a quinoline concentration of 5 mg L−1 were used for testing in the presence of substrate sufficient conditions, and the NAA used were 68.6 ± 1.7%, 57.1 ± 12.5%, 88.3 ± 3.7% and 49.4 ± 4.5%, respectively. The NAA decreased as the exposure time increased (apart from 48 h). Short exposure time had little impact in the presence of starvation conditions. The microbial effect of pollutants was related to the microbial metabolism. Compared with starvation conditions, there was a stronger inhibitory influence
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NO2−-N conversion to NH4+-N concentration (RS) of 1.11 ± 0.12 and of NO3−-N production to NH4+-N consumption (RP) of 0.18 ± 0.02 were both obtained. The RS and RP deviated from the theoretical values reported in other studies but were within normal ranges (Fernandes et al., 2018; Zhang et al., 2018).
− Fig. 2. Short-term effect of the initial NH+ 4 -N/NO2 -N concentration on the specific anammox activity (SAA) with/without quinoline.
due to exposure to Cu2+ in the presence of substrate (Chen et al., 2016). In this study, the effect of quinoline on anammox was not obvious due to the lower diffusion rate in granule sludge during shorter exposure times, and the suppression of SAA by quinoline was enhanced by starvation at longer exposure times. The SAA decreased sharply in the presence of quinoline and nitrite as exposure time increased. The NAA at different exposure times was 64.5 ± 1.5%, 52.6 ± 0.4%, 25.8 ± 6.9% and 6.2 ± 2.8%. Compared with the NAA in the presence of substrate sufficient conditions, the coexistence of NO2−-N and quinoline would aggravate the inhibition on anammox. 3.2. Long-term effects of quinoline on anammox performance 3.2.1. Performance of R1 without quinoline during the stabilizing phase (P0) During days 1–53 (P0), the influent of R1 was pumped without the addition of quinoline (Fig. 4). The R1 had a stable performance during this phase, and the nitrogen loading rate (NLR), nitrogen removal rate (NRR) and nitrogen removal efficiency (NRE) were 2.91 ± 0.24 kg m−3 d−1, 2.64 ± 0.10 kg m−3 d−1 and 91.24 ± 6.8%, respectively. As shown in Fig. 4, in the effluent, the NH4+-N and NO2−-N were 34.4 ± 19.4 mg L−1 and 12.4 ± 6.9 mg L−1, respectively, and with a shorter HRT, the reactor achieved a high removal capacity. Ratios of
Fig. 3. Effect of 5 mg L−1 quinoline on NAA in the presence of various levels of substrate.
3.2.2. Performance of R1 with high quinoline-induced stress (P1) During P1 (days 54–95), the anammox process was observed at different quinoline levels. Within three days in the presence of 1 mg L−1 quinoline, the NRE remained at 97.5 ± 2.4%. The performance of reactor was stable in this case. During days 57–75, the NRR and NRE of R1 were 2.8 ± 0.2 kg m−3 d−1 and 94.9 ± 2.3%, respectively, in the presence of 5 mg L−1 quinoline. During days 76–86, the NO2−-N of the effluent increased sharply from 27.2 to 71.4 mg L−1 in the presence of a quinoline concentration of 10 mg L−1, and the NRR continued to decrease until it reached 2.1 kg m−3 d−1. The RS and RP were 0.95 ± 0.11 and 0.15 ± 0.02, respectively, on days 57–75, and the abnormal reaction ratio indicated that the metabolic process of anammox was disturbed by the presence of quinoline. To recover the performance of reactor, the quinoline concentration in the influent was decreased to 5 mg L−1. However, the performance of the nitrogen removal process continued to deteriorate. The NH4+-N and NO2−-N concentrations in the effluent increased to 102.5 and 138.8 mg L−1, respectively, and the minimal NRR was 1.6 kg m−3 d−1. This result showed that anammox performance could not be restored, even if subjected to only a short period of quinoline-induced stress. The degree of disorder of RS decreased to 0.87 ± 0.12, and the RP was maintained at 0.15 ± 0.02. It is reasonable to assume that the accumulation of quinoline caused metabolic disorder. 3.2.3. Performance of R1 during the recovery phase (P2) On day 96, to accelerate the restoration of anammox performance, the NH4+-N/NO2−-N and quinoline concentrations in the influent were decreased to 210 and 0 mg L−1, respectively. The performance of R1 was gradually restored, and the effluent NO2−-N concentration was decreased to 18 mg L−1. Subsequently, the influent NH4+-N and NO2−-N concentrations were increased to 280 mg L−1, and the function of reactor stabilized on day 116. The average effluent NO2−-N and NRE were 33.3 ± 12.5 mg L−1 and 84.0 ± 4.9%, respectively, during days 116–129. The performance of R1 still had not fully recovered compared to that of P0 and P1, even though the NO2−-N concentration in the effluent was far below 70 mg L−1. After discontinuation of quinoline addition, RS and RP recovered to 1.02 ± 0.11 and 0.16 ± 0.04, respectively. The performance of the nitrogen removal process and the reaction ratio also recovered simultaneously after long-term acclimatization during this phase. 3.2.4. Performance of R1 after acclimatization with various levels of quinoline (P3) The resistance of anammox bacteria to quinoline was enhanced via sludge acclimatization. As shown in Table 2, the influent quinoline concentration was gradually increased from 1 to 5 mg L−1, and the NRR and NRE of R1 were 2.7 ± 0.2 kg m−3 d−1 and 92.1 ± 4.3%, respectively. Although the influent contained quinoline, the performance of the reactor was gradually restored. The NRR was stabilized in the presence of 10 mg L−1 quinoline within 12 days. After long-term acclimatization, anammox constituents had certain ability to tolerate quinoline. The degrees of disorder of RS and RP (1.05 ± 0.15 and 0.23 ± 0.09), also decreased. Taking what is above-mentioned into account, it could provide new insights into the operation of anammox processes for treating wastewater containing quinoline. Pre-treatment is necessary to control quinoline concentration in a proper range. And gradual acclimatization is also a crucial step to treat coking wastewater according to our longterm effects experiments. What's more, water quality monitoring and early warning need to be intensified when necessary.
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− − Fig. 4. Performance of the anammox reactor. A, the concentration of NH+ 4 -N, NO2 -N and NO3 -N in the influent and effluent; B, the NLR, NRR and NRE of the anammox reactor in the presence of different concentrations of quinoline.
3.3. Long-term effects of quinoline on the properties of anammox granules 3.3.1. SAA The SAA and biomass co-determined the anammox performance (Tsushima et al., 2007). As is depicted in Fig. 5, the SAA had a various
response under different quinoline concentration. Critical points were selected at which to evaluate the SAA in the continuous flow reactor. The SAA of inoculated sludge was 176.7 ± 16.1 mg N g−1 VSS d−1. On day 77, the SAA decreased to 127.6 ± 4.5 mg N g−1 VSS d−1 in the presence of 5 mg L−1 quinoline, and the percent of loss was 27.8%. This result
Table 2 Testing conditions and performance of the anammox reactor during different operational phases. Phase
Experimental aim
Operation time
− NH+ 4 -N/NO2 -N concentration (mg L−1)
Quinoline concentration (mg L−1)
NRE: (%)
NLR: (kg m−3 d−1)
NRR: (kg m−3 d−1)
P0 P1
Performance without quinoline Performance with increasing quinoline quickly
P2 P3
Performance recovery without quinoline Performance with increasing quinoline slowly
1–53 d 54–56 d 57–75 d 76–84 d 85–95 d 96–138 d 139–152 d 153–166 d 167–180 d 181–192 d 193–204 d 205–219 d 220–232 d
280 280 280 280 280 140,210,280 280 280 280 280 280 280 280
0 1 5 10 5 0 1 2 3 4 5 8 10
92.2 ± 3.8 97.5 ± 2.4 94.9 ± 2.3 76.1 ± 10.5 63.5 ± 6.4 80.1 ± 10.0 85.0 ± 10.4 91.0 ± 3.5 91.4 ± 4.3 94.2 ± 1.5 97.1 ± 2.7 89.8 ± 6.1 92.2 ± 5.3
2.8 ± 0.2 2.4 ± 0.0 2.9 ± 0.2 2.8 ± 0.2 2.8 ± 0.2 2.4 ± 0.5 2.8 ± 0.2 2.8 ± 0.2 3.0 ± 0.2 2.8 ± 0.1 2.6 ± 0.5 2.5 ± 0.6 2.6 ± 0.3
2.6 ± 0.2 2.3 ± 0.0 2.8 ± 0.2 2.1 ± 0.4 1.8 ± 0.2 1.9 ± 0.5 2.3 ± 0.3 2.5 ± 0.3 2.8 ± 0.2 2.7 ± 0.1 2.6 ± 0.4 2.3 ± 0.6 2.4 ± 0.3
NRE: nitrogen removal efficiency; NLR: nitrogen loading rate; NRR: nitrogen removal rate.
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demonstrated that anammox was suppressed by a low concentration of quinoline. As the reactor was in a state of functional redundancy, the NRR or NLR had changed very little. Therefore, the removal of nitrogen was not obviously affected when accompanied with a decrease in SAA. There was a persistent effect of quinolone on the activity of the destroyed anammox granules. Thus, anammox performance and bioactivity did not achieve an immediate recovery after removal of quinoline from the influent. The minimal SAA was 29.7 ± 7.5 mg N g−1 VSS d−1 without quinoline in P2 (day 111). In P3 (days 139–232), a strategy of gradually increasing the concentration was used to cultivate and acclimate the granules to low concentrations of quinoline. The performance and SAA were still gradually increased, though 1 mg L−1 quinoline was added again on day 129. The SAA recovered to 97.3% of the initial level after long-term operation of the reactor (day 220). The influent quinoline concentration was increased to 10 mg L−1, and the reactor demonstrated stable performance. Therefore, the reactor was able to tolerate 10 mg L−1 quinoline after a long-term acclimatization. 3.3.2. EPS and settling velocity EPS plays a crucial role in the resistance of toxic substances. EPS was secreted at a higher level by the microbial cells in activated sludge, biogranules and biofilms in order to provide for selfdefense from toxic substances such as heavy metals (Li and Yu, 2014). Zhang et al. (2015) regarded EPS as a distinct self-response to Cu(II) stress. The level of EPS (PN and PS) is shown in Fig. 4.6A, and the trend of its production during the entire test was first of decrease and then increase. The EPS content decreased by 49.3% compared to that obtained without quinoline accretion on day 152. Later, the increased concentration of quinoline stimulated the production of EPS, so the EPS content gradually increased after day 152. At the end of the experiment, the level of EPS was back to 60.4% of the initial value. The changing trend of EPS production in this study was inconsistent with our previous study, where it showed an increase when anammox sludge was exposed to SCN− (Chen et al., 2017a). Xu et al. (2018) found the EPS content varied obviously under different shock tests. There are many factors may affect the content of EPS, such as the microbial community structure, temperature, substrate (Nichols et al., 2004). Therefore, the trend of EPS content in this study needs a further exploration. A suitable settling velocity has been associated with sludge retention. In this study, the initial settling velocity was 63.9 ± 15.9 m h−1, and the value had increased to 71.3 ± 8.2 m h−1 by day 129. The PN/ PS of EPS was in line with the settling velocity and intensity (Franco et al., 2006; Tang et al., 2011; Wu et al., 2009). Franco et al. (2006) found that anammox granule sludge possessed a better settleability
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when it had a relatively low PN/PS ratio. On day 167, the minimum PN/PS ratio was 7.1, with a maximum settleability of 72.1 ± 12.1 m h−1. Interestingly, an opposing trend was observed in that the settling velocity increased with the PN/PS ratio, increasing after day 129. The maximum of PN/PS ratio was 19.5 on day 129, with a settleability of 70.7 ± 11.5 m h−1. The degree of the relationship between PN/PS and sludge settleability warrants further investigation. 3.3.3. Heme c Heme is an essential part of the pivotal anammox enzymes, including hydrazine synthesis (HZS), hydroxylamine oxidoreductase (HAO), and hydrazine oxidase (HZO) (Jetten et al., 2009). Anammox bacteria are rich in heme, which is a vital cofactor of cytochrome enzymes as it is involved in energy metabolism. Heme c is hypothesized to play an essential role in the carmine color of the anammox sludge, and the content of heme c might be related to bioactivity (Franco et al., 2006). During the course of the entire experiment, the trend of the change of the heme c level was consistent with that of the SAA. The heme c level had fallen only slightly from the initial value due to the addition of quinoline, from 3.9 ± 0.02 to 3.8 ± 0.04 μmol g−1 VSS, which had the synchronous trend with SAA by day 81. And the loss of SAA was 54.2% at that time. This result indicated that the anammox activity was enormously impacted by the presence of quinoline inhibition. The level of heme c had decreased to 1.3 ± 0.02 μmol g−1 VSS reaching the minimum value on day 129. Furthermore, SAA recovered to some extent from that moment on. On day 208, the heme c level had recovered to 3.1 ± 0.5 μmol g−1 VSS. Heme c content is related to NRR and SAA (Tang et al., 2011). Xu et al. (2019) demonstrated that the variation trend of heme c content in the anammox system was synchronous with NRR and SAA under NiO NPs exposure. In this study, the recovery of heme c content was slower than that of the SAA. More recently, a similar phenomenon was observed in a SCN− shock test, and the restoration time of heme c was considerably longer than that of SAA (Chen et al., 2017b). 3.3.4. Granule diameter The performance of the anammox granules is an essential factor affecting the reactor process. The granule diameter is dependent upon many factors, such as the reactor configuration, substrate loading rate, solid retention time, HRT, temperature, and pH (Marshall et al., 1984). The specific surface area of particle isn't too high to ensure the resistance to the load and hydraulic shocks (Yang and Jin, 2013). In this study, the sludge diameters determined under various quinoline concentrations are shown in Fig. 6D. In the whole anammox operation, the diameters of the anammox granules varied from 2.6 ± 0.7 to 2.3 ± 0.6 mm, with an average value of 2.4 ± 0.7 mm. In the initial 152 days, the granules diameter decreased from 2.6 ± 0.7 to 2.0 ± 0.52 mm due to the addition of quinoline. This result indicated that the quinoline presented in the reactor may partially destroy the granule structure. After 152 days, the diameter was 2.24 ± 0.6 mm and was hardly changed in the presence of quinoline. On day 208, the diameter was 76.9% of the initial value, and nearly 95% of the anammox granules had diameters of less than 2.5 mm. 4. Conclusion
Fig. 5. The SAA of anammox granules in different phases.
The short-term effects of quinoline on anammox were assessed based on quinoline concentration, substrate level, pre-exposure time and the presence of starvation conditions. The inhibition of anammox by quinoline was non-competitive, and the IC50 of quinoline on anammox mixed culture was 13.07 mg L−1. The anammox consortia could tolerate 10 mg L−1 quinoline by means of the gradient acclimatization during the continuous assays. The levels of EPS and heme c and granule diameter decreased by 49.3%, 67.1% and 23.2%, respectively, after inhibition with quinoline. These results may have a guiding role
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Fig. 6. The characteristics of anammox granules at different phases. A, EPS amounts and compositions; B, Heme c levels; C, Settling velocity; D, Diameter.
in propelling the application of the anammox process to treat wastewater containing quinoline.
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