- Email: [email protected]
Response of nitrogen removal performance, functional genes abundances and N-acyl-homoserine lactones release to carminic acid of anammox biomass
Bioresource Technology 299 (2020) 122567
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Response of nitrogen removal performance, functional genes abundances and N-acyl-homoserine lactones release to carminic acid of anammox biomass
T
⁎
Lingjie Liu, Min Ji, Fen Wang , Zhao Yan, Zhongke Tian School of Environmental Science and Engineering, Tianjin University, Tianjin 300350, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Carminic acid Anammox N-acyl-homoserine lactones Quantitative reverse transcription polymerase chain reaction
Carminic acid (CA) can serve as a redox mediator and influence the electron transfer process. CA dosages of 0–5 mg/L were added to anaerobic ammonia oxidation (anammox) biomass. The results illustrated that CA not only reduced the inorganic nitrogen removal efficiency, but also decreased the nitrogen removal rate. The deterioration of nitrogen removal performance was due to the excess production of nitrate-nitrogen. The concentration of extracellular polymeric substances showed a decrease together with a decline in N-acyl-homoserine lactones release. CA addition decreased the activity of anammox bacteria while increasing the nitrifying potential. Quantitative reverse transcription polymerase chain reaction showed a decrease in anammox functional genes (nirS, hzo, and hzsB) and promotion of the expression of the nxrB gene, which corresponded with a decrease in anammox bacteria activity and the improvement of nitrifying potential. As a result, CA should not be added to anammox biomass.
1. Introduction The anaerobic ammonia oxidation (anammox) process is a biological nitrogen removal process by which ammonium-nitrogen (NH+4 - N ) is directly transferred to nitrogen gas using nitrite-nitrogen (NO−2 - N ) as
⁎
the electron acceptor (Jetten et al., 2001). Compared with traditional biological nitrogen removal technologies (Klein et al., 2017a; Klein et al., 2017b), anammox is recognized as a sustainable and environmentally friendly process for inorganic nitrogen removal in wastewater treatment because the process does not require oxygen or
Corresponding author at: Peiyang Park Campus: No. 135 Yaguan Road, Haihe Education Park, 300350 Tianjin, China. E-mail address: [email protected] (F. Wang).
https://doi.org/10.1016/j.biortech.2019.122567 Received 30 September 2019; Received in revised form 2 December 2019; Accepted 4 December 2019 Available online 07 December 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
2. Materials and methods
organic carbon and reduces the carbon dioxide and nitrous oxide emissions (Lackner et al., 2014; Rikmann et al., 2018; Zekker et al., 2019). This process can reduce the cost of wastewater treatment (Rikmann et al., 2018; Zekker et al., 2019). A redox mediator is a molecule that can function as an electron shuttle between electron acceptors and bacteria (Rabaey et al., 2005). Electron shuttles have the ability of transferring electrons between an extensive variety of compounds in redox reactions, including inorganic and organic compounds (Van der Zee and Cervantes, 2009). Various redox mediators have been applied in wastewater treatment, especially the microbial electron transfer process (Van der Zee and Cervantes, 2009). Qiao et al. (2014) investigated the short-term effect of three redox mediators (anthraquinone-2,6-disulfonate, 2-hydroxy-1,4-naphthoquinone, and anthraquinone-2-carboxylic acid) on the anammox process, and the results showed that the increase in the dosage of redox mediators had a more negative impact on nitrogen removal. Rikmann et al. (2014) also reported that anthraquinone-2,6-disulfonate improved the production of hydrazine (N2H4) and that the total nitrogen removal rate was further increased by 20%. Graphene oxide had been reported as an electron shuttle to increase the redox conversion process (Colunga et al., 2015). In the study conducted by Wang et al. (2013), a maximum increase of 10.26% in anammox activity together with greater extracellular polymeric substances (EPS) production was obtained with 0.1 g/L of graphene oxide. Furthermore, reduced graphene oxide was reported to have a greater ability of electron transfer than graphene oxide by approximately three orders of magnitude, with the total nitrogen removal rate and enzyme activity increasing by 10.2% and 1.5–2 fold, respectively (Wang et al., 2013). Quorum sensing (QS) refers to the cell-cell communication process of the ability of bacteria to sense their density by exchanging communication molecules called autoinducers (Jemielita et al., 2018). Recently, the metabolic pathways and communication mechanism of Nacyl-homoserine lactones (AHLs)-mediated QS regulation have been mentioned in the anammox consortia (Tang et al., 2018a; Tang et al., 2018b). In particular, N-(3-oxohexanoyl)-DL-homoserine lactone (3oxo-C6-HSL), N-hexanoyl-DL-homoserine lactone (C6-HSL), and N-octanoyl-DL-homoserine lactone (C8-HSL) regulate the electron transport carriers that influence bacterial activity (Tang et al., 2018b). Adding Ndodecanoyl-DL-homoserine lactone (C12-HSL) into the reactors can reduce the anammox process start-up period to 66 d compared with the control group start-up period of 80 d, thereby indicating that C12-HSL has the ability to promote the specific anammox activity (Zhao et al., 2018). Carminic acid (CA) is a deep red anthraquinone that is extracted from scale insects, and it is widely used as a pigment in different fields, e.g., crimson ink, paints, cosmetics, and food coloring (Dapson, 2007). CA has the redox group of anthraquinone and the oxidation group of hydroquinone, and it can act as a redox mediator (Li et al., 2013). Wolf et al. (2009) explored the effects of quinones containing various redox functional groups on microbial ferrihydrite reduction kinetics by Geobacter metallireducens at low concentrations, and found that CA concentrations of 0.1–10.0 μm had no apparent effect on the iron reduction kinetics. Li et al. (2013) also investigated the effects of CA on goethite reduction and current production by Klebsiella pneumonia L17, and the result showed that there was no clear effect on Fe(III) reduction. To date, study on CA addition to anammox biomass was lacking. In this study, different concentrations of exogenous CA were added to anammox biomass, and the following aspects were determined: (1) the response of nitrogen removal performance, (2) the change in QS signal molecule concentrations and EPS concentrations, and (3) the variation of anammox bacteria activity, nitrifying potential, and the change in functional genes expression activities according to quantitative reverse transcription polymerase chain reaction (qRT-PCR) after CA addition.
2.1. Original anammox biomass and CA The anammox biomass was obtained from a 4 L biofilm reactor that had been operating in a laboratory for 1.5 years with a stable total inorganic nitrogen (TIN) removal efficiency of more than 80%, with 100 mg/L of NH+4 - N and 132 mg/L of NO−2 - N in the influent under 35 ± 1 ℃. The hydraulic retention time was maintained at 24 h. CA (CAS No. 1260-17-9) was purchased from Dr. Ehrenstorfer GmbH (Germany). 2.2. Set up of batch experiments Serum flasks with an effective volume of 250 mL were used for batch exposure assays, and the volatile suspended solids (VSS) concentration was 2200 mg/L in each flask. Before feeding into the flask, anammox biomass was washed in phosphate-buffered saline (PBS) (0.14 g/L of KH2PO4 and 0.75 g/L of K2HPO4) three times in order to eliminate the residual nitrogen in the biomass. The synthetic wastewater contained 50 mg N/L of (NH4)2SO4 and 50 mg N/L of Na2NO2 as the ammonia and nitrite sources, respectively, 500 mg/L of NaHCO3 for alkalinity addition, and 100 mg/L of MgSO4∙7H2O, 180 mg/L of CaCl2⋅2H2O, and 27 mg/L of KH2PO4. In addition, 1 mL/L of trace element solutions Ⅰ and Ⅱ were added to the influent (Sliekers et al., 2002). The liquid was purged with highly purified nitrogen gas for at least 10 min to remove dissolved oxygen (DO). Six sets of batch experiments were conducted with CA concentrations of 0, 0.05, 0.1, 0.5, 1, and 5 mg/L, and each set of experiments was conducted in triplicate. The control group was set as 0 mg/L of CA addition. The original pH was fixed at 7.8 by 1 M HCl and 1 M NaOH. The operating temperature of the shaker was maintained at 37 ± 0.2 ℃ and the shaker speed was 170 rpm to maintain full contact. Liquid samples were gathered by a syringe with a long needle and periodically purged through 0.45 μm membranes for measurement of nitrogen species. 2.3. Analytical methods The concentration of nitrate-nitrogen (NO−3 - N ) was determined by ion-exchange chromatography (IC-1100, Thermo Fisher, USA). NH+4 - N , NO−2 - N , mixed liquor suspended solids, and VSS concentrations were detected based on the APHA Standard Methods (APHA, 2005). The concentration of TIN was calculated as the sum of NH+4 - N , NO−2 - N , and NO−3 - N . DO and pH were determined by a portable DO meter (HQ 30d, HACH, USA) and a digital pH meter (JENCO Model 6010, China), respectively. 2.4. Determination of anammox bacteria activity and nitrifying potential Anammox biomass was gathered at the end of 48 h to test the anammox bacteria activity and nitrifying potential. The anammox activity test was described by Dapena-Mora et al. (2010). Briefly, biomass samples were washed with PBS solution three times, and substrates of 20 mg/L of NH+4 - N and 20 mg/L of NO−2 - N were added. Each serum bottle contained approximately 400 mg VSS/L of biomass and was stripped with nitrogen gas for 10 min. Supernatants were obtained at intervals of 30 min to measure the NH+4 - N and NO−2 - N concentrations in order to calculate the anammox bacteria activity. This test was conducted in duplicate and the average value and standard deviation were calculated for analysis. For the nitrifying potential test, 20 mg/L of NH+4 - N or NO−2 - N was fed separately with adequate aeration. The liquid sampling interval was 30 min (Rich et al., 2008; Sun et al., 2019) and the concentrations of NH+4 - N , NO−2 - N , and NO−3 - N were detected to test the nitrifying potential (Almstrand et al., 2011). 2
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
NH+4 - N and NO−2 - N concentrations at the end of the reaction. However, NO−3 - N production was simulated under different CA dosages. When the reaction finished, the NO−3 - N concentrations of different CA dosages showed an increase from 13.40 mg/L to 31.07 mg/L (Fig. 1c). The nitrogen removal loading rates were 44.5, 38.6, 38.4, 37.8, 37.1, and 34.2 mg/(L·d), respectively, with CA dosages of 0–5 mg/L. The TIN removal efficiencies showed a decrease from 79.47% to 61.13% with the increase in CA dosages from 0 mg/L to 5 mg/L, respectively. In addition, the stoichiometric coefficient of ∆NH+4 - N:∆NO−3 -N described by Strous et al. (1998) was 0.26. The values of ∆NH+4 - N:∆NO−3 -N were 0.26, 0.50, 0.51, 0.53, 0.56, and 0.66 (p less than 0.01) with CA dosages from 0 mg/L to 5 mg/L. It was clear that the generation of NO−3 - N was greater than the theoretical value after the addition of CA. The decrease in TIN removal efficiency in this study was due to the excess production of NO−3 - N . The TIN degradation in this study followed a zero-order reaction kinetic model. Parameters of the zero-order kinetic model were summarized in Table 1. The results demonstrated that the TIN removal rate of the control group of 1.96 mg N/(L·h) was higher than those of the groups with CA addition, which were 1.71 mg N/(L·h), 1.70 mg N/(L·h), 1.64 mg N/(L·h), 1.62 mg N/(L·h), and 1.44 mg N/(L·h), respectively. The results indicated that CA reduced the nitrogen removal rate. It was clear that the presence of a certain amount of CA not only increased the NO−3 - N production, which resulted in the reduction in TIN removal efficiency, but also decreased the inorganic nitrogen reduction rate.
2.5. Extracellular polymeric substances extraction and analysis Biomass samples were gathered after the experiment was finished (48 h) for EPS analysis. A modified method with heat extraction was utilized for EPS extraction, and polysaccharides (PS) and proteins (PN) were determined by the Lowry-Folin method (Tan et al., 1984) using bovine serum albumin (Sigma-Aldrich) as the standard and by the phenol–sulfuric acid method using glucose (Yuanli Co., Ltd., Tianjin, China) as the standard (Nielsen, 2010), respectively. 2.6. AHLs extraction and measurement Standard AHLs (> 97%, HPLC), including C8-HSL, C12-HSL, and N(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-HSL), were purchased from Sigma-Aldrich. The AHLs extraction procedures from the water and biomass phases were described by Sun et al. (2019), and some modification was made. Briefly, at the end of the exposure experiment, 100 mL of supernatant was filtered through 0.45 μm membranes and concentrated by solidphase extraction. After drying for 30 min and elution with 5 mL of acetonitrile, liquid samples were evaporated to dryness with nitrogen gas and further re-dissolved in 0.1 mL of acetonitrile for UPLC-MS/MS (Waters, USA) analysis. As for the AHLs extraction in biomass, 25 mL sludge samples were collected and centrifuged for 5 min at 4000 rpm to remove the supernatant. After freeze-drying for 36 h at −80 ℃, 10 mL of ethyl acetate was added to extract the AHLs by ultrasonic extraction (400 W, 15 min; KQ-300DE, Kunshan, China) and a vortex process. This pretreatment was repeated three times. The mixers were conducted at 4000 rpm for 15 min and supernatant was gathered. Further steps were the same as those for the water phase mentioned above.
3.2. Effect of CA addition on anammox bacteria activity and nitrifying potential The microbial activity of anammox bacteria and the nitrifying potential under different CA dosages were illustrated in Fig. 2a and b, respectively. The activity of anammox bacteria showed a decrease with CA addition from 0 mg/L to 5 mg/L. The anammox bacteria activity in the control group was 3.73 mg N/g VSS, which was 1.96 times higher than that of the group with 5 mg/L of CA addition. The exogenous CA dosages of 0.05 mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L and 5 mg/L decreased the anammox bacteria activities by 26%, 34%, 41%, 44%, and 49%, respectively. As for the nitrifying potential, the removal rates of NH+4 - N and − NO2 - N were approximately 0.20 mg N/g VSS and 2.00 mg N/g VSS, respectively, with 0.1 mg/L of CA addition, while they increased to 0.48 mg N/g VSS and 2.78 mg N/g VSS, respectively, with a CA dosage of 5 mg/L. In contrast with the anammox bacteria activity, the nitrifying potential showed an increasing trend with the increase in CA addition. As reported in a previous study, some exogenous quinones would cause cell toxicity because of toxic organic compounds (Bolton and Dunlap, 2016). In addition, the activities of enzymes, such as nitrite reductase and hydrazine dehydrogenase, are inhibited with the addition of more than 0.6 mM 2-hydroxy-1,4-naphthoquinone (Qiao et al., 2014). Ahn (2006) observed a significant correlation between the nitrogen removal rate and microbial activity. Previous studies indicated that the nitrogen removal rate or nitrogen removal efficiency decreased in the anammox process together with the reduction in anammox activity (Jin et al., 2013). In this study, the decrease in anammox bacteria activity was accompanied by a downward trend in the NO+4 - N and NO−2 - N removal rates. The increased concentrations of NO−3 - N at the end of the experiment were attributed to the enhancement of the nitrifying potential, which corresponded with the study conducted by Poot et al. (2016). This led to a decrease in TIN removal efficiency.
2.7. RNA isolation, reverse transcription, and qRT-PCR analysis Biomass samples were collected at the end of 48 h. After the samples were centrifuged at 10 000 rpm for 5 min, a Direct-zol RNA MiniPrep kit was used for RNA extraction (Zymo Research, CA). The RNA concentrations were measured using a Nanodrop spectrophotometer (Quawell UV–vis spectrophotometer Q5000, USA). In addition, RNA was used for cDNA synthesis by reversing the transcription process using the HiScript Ⅱ 1st Strand cDNA Synthesis Kit (Vazyme). The detailed process of establishing clone libraries is presented in the Supplementary Data. qRT-PCR was conducted by an iQ5 Bio-Rad thermal cycler adopting SYBR green chemistry. Each reaction mixture included 2 × TB Green Premix EX Taq Ⅱ (Takara) (12.5 μL), forward primer (10 mM, 1 μL), reverse primer (10 mM, 1 μL), template cDNA (1 μL) and sterile deionized water (9.5 μL). The PCR program was initiated with denaturation for 2 min at 95 ℃, followed by 40 cycles of 5 s at 95 ℃, 60 s at the annealing temperature, and 30 s at 72 ℃. The primers for qRT-PCR are summarized in the Supplementary Data. Triplicate data assays were conducted using properly diluted samples and negative controls (Bustin and Nolan, 2004). In addition, the relative abundance of functional gene expression was analyzed using the 2−ΔΔCt method, and error bars represented the standard errors (Lin et al., 2016). 3. Results and discussion 3.1. Response of nitrogen removal performance to CA addition Fig. 1 illustrated the short-term response of nitrogen removal performance to CA addition in the anammox system. During the batch tests, the nitrogen removal performance of experimental groups worsened compared with that of the control group. After 48 h of exposure, the concentrations of NH+4 - N and NO−2 - N were maintained at approximately 10 mg/L and 0 mg/L, respectively (Fig. 1a and b). CA presented in the anammox system had no significant impact on the
3.3. Variation of the amount and composition of EPS EPS, which are mainly secreted from microbial organisms, produced by cellular lysis, and hydrolyzed by macromolecules, are distributed both outside cells and inside microbial aggregates (Sheng et al., 2010). 3
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
Fig. 1. NH+4 - N (a), NO−2 - N (b), NO− 3 - N (c) concentrations and the TIN removal efficiencies (d) under different CA doses with influent TIN loading rate of 55.95 mg/ (L·d).
the decrease in EPS concentration was associated with the increase in CA concentration. The EPS concentration decreased by 5.88% at 0.05 mg/L of CA addition, and further decreased by 31.46% at 5 mg/L of CA addition. Previous studies demonstrated that more EPS secretion had a positive effect on nitrogen removal performance (Miao et al., 2018). Zhang et al. (2017) thought that the reduction in PN from 271.5 mg/g VSS to 218.7 mg/g VSS might result from the suppressed metabolic activity of anammox under metallic nanoparticle stress. Similarly, the reduction in EPS occurred with the decrease in TIN removal efficiency in this study. The ratio of PN/PS reflected the surface hydrophobicity of the sludge (Yan et al., 2015). When the ratio increased, the surface properties of the sludge could effectively change, thereby contributing to the cohesion between the aggregates and the tightness and stability of the structure (McSwain et al., 2005). The ratios of PN/PS with CA dosages
Table 1 Kinetic parameters of TIN removal under different CA doses. CA doses (mg/L)
K0 (mg N/(L·h))
R2
0 0.05 0.1 0.5 1 5
1.96 1.71 1.70 1.64 1.62 1.44
0.9815 0.9820 0.9774 0.9778 0.9594 0.9401
PN and PS are the dominant components of EPS (Sheng et al., 2010). As shown in Fig. 3a, the concentrations of EPS were 77.21 mg/g VSS, 72.00 mg/g VSS, 63.71 mg/g VSS, 60.20 mg/g VSS, 56.18 mg/g VSS, and 52.43 mg/g VSS under different CA dosages. It could be seen that
Fig. 2. Variation of anammox bacteria activities (a), nitrifying potential (b) under different CA doses. 4
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
Fig. 3. EPS concentrations (a) and PN/PS (b) under different CA addition.
from 0 mg/L to 5 mg/L were 3.85, 2.43, 2.07, 2.25, 2.09, and 2.00. There was a decrease in PN/PS as the CA concentration increased in the anammox system. The addition of CA in the anammox system might have caused the deterioration of the sludge stability. 3.4. Variation of AHLs release under CA addition QS can orchestrate gene expression programs among bacteria and further underlie collective activities (Papenfort and Bassler, 2016). It has been proven that Candidatus Kuenenia, a prevailing genus in the anammox process (He et al., 2018), contains hdtS, which is a type of signal molecule synthesis gene, and has the potential to synthesize AHLs (Sun et al., 2019). C8-HSL, C12-HSL, and 3-oxo-C8-HSL were detected in both the water and biomass phases in this experiment (Fig. 4). The dominant QS signal molecules were C8-HSL and C12-HSL in the water and biomass phases, respectively. There was an apparent decrease in the C8-HSL and C12-HSL concentrations in the water phase from 6.93 ng/L to 2.48 ng/L and from 3.45 ng/L to 0.38 ng/L, respectively, with CA addition from 0 mg/L to 5 mg/L. The concentration of 3-oxo-C8-HSL was maintained at approximately 0.88 ng/L when the CA addition was less than 0.5 mg/ L, and then showed a slight decrease to 0.54 ng/L with a CA dosage of 5 mg/L. Similarly, the QS signals in the biomass also demonstrated an overall decreasing trend. The concentrations of C12-HSL were 46.98 ng/g VSS, 22.43 ng/g VSS, 23.91 ng/g VSS, 15.26 ng/g VSS, 14.56 ng/g VSS, and 7.36 ng/g VSS with CA addition from 0 mg/L to 5 mg/L. The C8-HSL concentration was reduced by 40.3% with 0.05 mg/L of CA addition and then decreased by 67.8% with 5 mg/L of CA addition. The concentration of 3-oxo-C8-HSL also showed a slight decrease from 21.68 ng/L to 15.55 ng/L with CA dosages from 0 mg/L to 5 mg/L, respectively. Thus, CA had a negative effect on QS in the anammox system, especially for C8-HSL and C12-HSL in both the water and biomass phases. It had been reported by Li et al. (2014) that the adsorption peaks of CA by the FTIR spectrum were at 3366 cm−1 and 1719 cm−1, which illustrated OeH and C]O in the carboxylic group, respectively. In the study conducted by Wolf et al. (2009), it was noted that CA had no activity or only minor activity of electron shuttling in theory because of the low redox potential. In addition, previous research considered that the redox potential of CA (-500 mV) was lower than −400 mV (the lowest value of c-cytochrome), which might explain its ineffective ability of acting as an exogenous electron shuttle for microbial Fe(III) reduction (Li et al., 2013). In addition, chronoamperometry technology demonstrated that the electron accepting capacity and donating capacity of CA were relatively low, and the ability of electron transfer of CA was weak (Li et al., 2013). In this study, CA addition reduced the rate of electron transfer and further weakened the communication among bacteria, thereby resulting in the decrease in AHLs release.
Fig. 4. The variation of AHLs in water (a) and biomass (b) phases under different CA addition.
The EPS concentrations appeared to decrease by 32.09% in this study corresponding with the decrease in QS signal concentrations. A similar result from Zhao et al. (2016) showed that porcine kidney acylase Ⅰ as well as vanillin could inactivate AHLs and lead to anammox activity reduction at an enzyme level. It had been reported that C6-HSL and C8-HSL were overproduced, especially when nitrite-oxidizing bacteria (NOB) activity was promoted (Mellbye et al., 2017). However, the C6-HSL and C8-HSL concentrations showed a decreasing trend while NO−3 - N showed an increasing trend in this study, and the specific reasons underlying this phenomenon needed to be confirmed.
5
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
Fig. 5. The effects of different doses of CA on the activity of functional genes by qRT-PCR, nirS (a), hzsB (b), hzo (c), nxrB (d) and ccsB (e).
showed a decline, and were approximately 0.24 fold and 0.05 fold greater than that of the control, respectively, with CA addition of 5 mg/ L. This result indicated that CA might restrain the anammox process in N2H4 generation. Furthermore, as reported previously, AHLs influenced the expression of hzsA, which regulated the anammox activity (Tang et al., 2019). Thus, the decreasing trend of AHLs concentrations regulated the anammox genes expression and controlled the activity of anammox to some extent. The nxrB gene is considered a phylogenetic marker and functional gene for Nitrospira, which could oxide NO−2 - N to NO−3 - N . Unlike the trend of anammox functional genes, the level of nxrB gene expression showed a slight increase from 4.96 fold to 9.78 fold greater than the control with 0.01–5 mg/L of CA addition. As described in Section 3.1, the concentration of NO−3 - N at the end of the batch experiments
3.5. Variation of the activity of functional genes by qRT-PCR under CA addition qRT-PCR technology is widely used in gene expression studies (Hu et al., 2016). To study the influence of CA addition on the anammox biomass at the transcriptional level, an expression assay of genes encoding NIR, HZS, HZO, NXR, and CCS was performed. The relative levels of different functional genes were normalized to the control group with a value of 1, and were shown in Fig. 5. The nirS gene, which is catalyzed by nitric oxide oxidoreductase (NirS), can reduce NO−2 - N to NO, which is the first step in anammox reaction. The expression of the nirS gene showed a slight decrease and was finally maintained at around 0.08 fold greater than that of the control group, which corresponded with the decrease in the NO−2 - N reduction rate. The expression of anammox-related functional genes, namely hzsB and hzo, also
6
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
References
increased to 31.07 mg/L, which was the main reason for the decrease in the TIN removal efficiency and corresponded with the higher level of nxrB gene expression. The chemical properties offered by heme are utilized in multiple PN that implement an array of functions, e.g., redox catalysis and electron transfer (Poulos, 2014). Heme c is covalently bound, and has the ability of catalysis of various redox reactions as well as electron transfer (Bertini et al., 2006). ccsB is one of the components of the cytochrome c synthase (Simon and Hederstedt, 2011). After CA addition from 0.01 mg/L to 5 mg/L, the expression of the ccsB gene showed a decreasing trend from 0.71 to 0.13 fold compared with control group, which indicated that enzyme synthesis of cytochrome c was repressed. Furthermore, the electron transport and communication among bacteria were reduced, which caused the deterioration of nitrogen removal performance. Overall, the qRT-PCR results revealed that the expression of anammox-related genes weakened and that of nitrification-related genes strengthened, which was in accordance with the variation of anammox activity and nitrifying potential. As a result, it led to the excess production of NO−3 - N and decreased TIN removal efficiency. The maintenance of anammox bacteria and suppression of NOB were the key issues for maintaining operational stability of the anammox process (Persson et al., 2014). It had been reported that the multiplication of NOB resulted in irreversible inhibition of the anammox system because of competition with NO−2 - N as an electron acceptor (Joss et al., 2011). In summary, in order to maintain the stability of the anammox system, CA should not be added to the system.
Ahn, Y.H., 2006. Sustainable nitrogen elimination biotechnologies: a review. Process. Biochem. 41, 1709–1721. Almstrand, R., Lydmark, P., Sörensson, F., Hermansson, M., 2011. Nitrification potential and population dynamics of nitrifying bacterial biofilms in response to controlled shifts of ammonium concentrations in wastewater trickling filters. Bioresour. Technol. 102, 7685–7691. APHA, A., WEF, 2005. Standard methods for the examination of water and wastewater. 21 st ed. Washington, DC. Bertini, I., Cavallaro, G., Rosato, A., 2006. Cytochrome c: occurrence and functions. Chem. Rev. 106, 90–115. Bolton, J.L., Dunlap, T., 2016. Formation and biological targets of quinones: cytotoxic versus cytoprotective effects. Chem. Res. Toxicol. 30, 13–37. Bustin, S.A., Nolan, T., 2004. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Technol. 15, 155. Colunga, A., Rangel-Mendez, J.R., Celis, L.B., Cervantes, F.J., 2015. Graphene oxide as electron shuttle for increased redox conversion of contaminants under methanogenic and sulfate-reducing conditions. Bioresour. Technol. 175, 309–314. Dapena-Mora, A., Vázquez-Padín, J., Campos, J., Mosquera-Corral, A., Jetten, M., Méndez, R., 2010. Monitoring the stability of an Anammox reactor under high salinity conditions. Biochem. Eng. J. 51, 167–171. Dapson, R., 2007. The history, chemistry and modes of action of carmine and related dyes. Biotech. Histochem. 82, 173–187. He, S., Chen, Y., Qin, M., Mao, Z., Yuan, L., Niu, Q., Tan, X., 2018. Effects of temperature on anammox performance and community structure. Bioresour. Technol. 260, 186–195. Hu, Y., Deng, T., Chen, L., Wu, H., Zhang, S., 2016. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata. PLoS One 11, e0154384. Jemielita, M., Yan, J., Hurley, A., Stone, H., Wingreen, N., Bassler, B., 2018. Quorum Sensing Control of Vibrio Choleraeaggregation. APS Meeting Abstracts. Jetten, M.S., Wagner, M., Fuerst, J., van Loosdrecht, M., Kuenen, G., Strous, M., 2001. Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Curr. Opin. Biotechnol. 12, 283–288. Jin, R.C., Ma, C., Yu, J.J., 2013. Performance of an Anammox UASB reactor at high load and low ambient temperature. Chem. Eng. J. 232, 17–25. Joss, A., Derlon, N., Cyprien, C., Burger, S., Szivak, I., Traber, J., Siegrist, H., Morgenroth, E., 2011. Combined nitritation–anammox: advances in understanding process stability. Environ. Sci. Technol. 45, 9735–9742. Klein, K., Kattel, E., Goi, A., Kivi, A., Dulova, N., Saluste, A., Zekker, I., Trapido, M., Tenno, T., 2017a. Combined treatment of pyrogenic wastewater from oil shale retorting. Oil Shale 34, 82–96. Klein, K., Kivi, A., Dulova, N., Zekker, I., Mölder, E., Tenno, T., Trapido, M., Tenno, T., 2017b. A pilot study of three-stage biological–chemical treatment of landfill leachate applying continuous ferric sludge reuse in Fenton-like process. Clean Technol. Environ. Policy 19, 541–551. Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H., van Loosdrecht, M.C., 2014. Full-scale partial nitritation/anammox experiences–an application survey. Water Res. 55, 292–303. Li, B., Li, Z., Situ, B., Dai, Z., Liu, Q., Wang, Q., Gu, D., Zheng, L., 2014. Sensitive HIV-1 detection in a homogeneous solution based on an electrochemical molecular beacon coupled with a nafion–graphene composite film modified screen-printed carbon electrode. Biosens. Bioelectron. 52, 330–336. Li, X., Liu, L., Liu, T., Yuan, T., Zhang, W., Li, F., Zhou, S., Li, Y., 2013. Electron transfer capacity dependence of quinone-mediated Fe (III) reduction and current generation by Klebsiella pneumoniae L17. Chemosphere 92, 218–224. Lin, W.P., Xiong, G.P., Lin, Q., Chen, X.W., Zhang, L.Q., Shi, J.X., Ke, Q.F., Lin, J.H., 2016. Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury. J. Neuroinflamm. 13, 52. McSwain, B., Irvine, R., Hausner, M., Wilderer, P., 2005. Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Appl. Environ. Microbiol. 71, 1051–1057. Mellbye, B.L., Spieck, E., Bottomley, P.J., Sayavedra-Soto, L.A., 2017. Acyl-homoserine lactone production in nitrifying bacteria of the genera Nitrosospira, Nitrobacter, and Nitrospira identified via a survey of putative quorum-sensing genes. Appl. Environ. Microbiol. 83, e01540–17. Miao, L., Zhang, Q., Wang, S., Li, B., Wang, Z., Zhang, S., Zhang, M., Peng, Y., 2018. Characterization of EPS compositions and microbial community in an Anammox SBBR system treating landfill leachate. Bioresour. Technol. 249, 108–116. Nielsen, S.S., 2010. Phenol-sulfuric acid method for total carbohydrates. In: Heifelberg, D. (Ed.), Food Analysis Laboratory Manual, Second ed. Springer, New York, pp. 47–55. Papenfort, K., Bassler, B.L., 2016. Quorum sensing signal–response systems in Gram-negative bacteria. Nat. Rev. Microbiol. 14, 576. Persson, F., Sultana, R., Suarez, M., Hermansson, M., Plaza, E., Wilén, B.-M., 2014. Structure and composition of biofilm communities in a moving bed biofilm reactor for nitritation–anammox at low temperatures. Bioresour. Technol. 154, 267–273. Poot, V., Hoekstra, M., Geleijnse, M.A., van Loosdrecht, M.C., Pérez, J., 2016. Effects of the residual ammonium concentration on NOB repression during partial nitritation with granular sludge. Water Res. 106, 518–530. Poulos, T.L., 2014. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962. Qiao, S., Tian, T., Zhou, J., 2014. Effects of quinoid redox mediators on the activity of anammox biomass. Bioresour. Technol. 152, 116–123. Rabaey, K., Boon, N., Höfte, M., Verstraete, W., 2005. Microbial phenazine production enhances electron transfer in biofuel cells. Environ. Sci. Technol. 39, 3401–3408. Rich, J.J., Dale, O.R., Song, B., Ward, B.B., 2008. Anaerobic ammonium oxidation
4. Conclusion EPS appeared to decrease after CA addition to the anammox system. The AHLs concentrations also decreased, especially C8-HSL in the water phase and C12-HSL in the biomass phase. CA inhibited anammox bacteria activity by 49% and promoted the nitrifying potential to 2.78 mg N/g VSS. The qRT-PCR results indicated that the expression of anammox-related genes (nirS, hzsB, and hzo) was weakened and that of nitrification-related genes (nxrB) was strengthened. This led to the excess production of NO−3 - N and decrease TIN removal efficiency. Overall, CA addition should be avoided to maintain the stability of the anammox system. CRediT authorship contribution statement Lingjie Liu: Writing - original draft, Conceptualization, Methodology, Investigation, Data curation, Visualization, Formal analysis. Min Ji: Supervision. Fen Wang: Writing - review & editing, Supervision, Funding acquisition. Zhao Yan: Methodology. Zhongke Tian: Methodology. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment of China (2017ZX07106). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122567. 7
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
regulatory pathway of acyl-homoserine lactones based quorum sensing in anammox consortia. Environ. Sci. Technol. 52, 2206–2216. Tang, X., Guo, Y., Zhu, T., Tao, H., Liu, S., 2019. Identification of quorum sensing signal AHLs synthases in Candidatus Jettenia caeni and their roles in anammox activity. Chemosphere 225, 608–617. Van der Zee, F.P., Cervantes, F.J., 2009. Impact and application of electron shuttles on the redox (bio) transformation of contaminants: a review. Biotechnol. Adv. 27, 256–277. Wang, D., Wang, G., Zhang, G., Xu, X., Yang, F., 2013. Using graphene oxide to enhance the activity of anammox bacteria for nitrogen removal. Bioresour. Technol. 131, 527–530. Wolf, M., Kappler, A., Jiang, J., Meckenstock, R.U., 2009. Effects of humic substances and quinones at low concentrations on ferrihydrite reduction by Geobacter metallireducens. Environ. Sci. Technol. 43, 5679–5685. Yan, L., Liu, Y., Wen, Y., Ren, Y., Hao, G., Zhang, Y., 2015. Role and significance of extracellular polymeric substances from granular sludge for simultaneous removal of organic matter and ammonia nitrogen. Bioresour. Technol. 179, 460–466. Zekker, I., Kivirüüt, A., Rikmann, E., Mandel, A., Jaagura, M., Tenno, T., Artemchuk, O., Rubin, S., Tenno, T., 2019. Enhanced efficiency of nitritating-anammox sequencing batch reactor achieved at low decrease rates of oxidation–reduction potential. Environ. Eng. Sci. 36, 350–360. Zhang, Z.Z., Xu, J.J., Shi, Z.J., Cheng, Y.F., Ji, Z.Q., Deng, R., Jin, R.C., 2017. Short-term impacts of Cu, CuO, ZnO and Ag nanoparticles (NPs) on anammox sludge: CuNPs make a difference. Bioresour. Technol. 235, 281–291. Zhao, R., Zhang, H., Zhang, F., Yang, F., 2018. Fast start-up anammox process using Acylhomoserine lactones (AHLs) containing supernatant. J. Environ. Sci. 65, 127–132. Zhao, R., Zhang, H., Zou, X., Yang, F., 2016. Effects of Inhibiting Acylated Homoserine Lactones (AHLs) on Anammox Activity and Stability of Granules’. Curr. Microbiol. 73, 108–114.
(anammox) in Chesapeake Bay sediments. Microb. Ecol. 55, 311–320. Rikmann, E., Zekker, I., Tenno, T., Saluste, A., Tenno, T., 2018. Inoculum -free start-up of biofilm- and sludge-based deammonification systems in pilot scale. Int. J. Environ. Sci. Technol. 15, 133–148. Rikmann, E., Zekker, I., Tomingas, M., Vabamäe, P., Kroon, K., Saluste, A., Tenno, T., Menert, A., Loorits, L., dC Rubin, S.S., 2014. Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors. J. Biosci. Bioeng. 118, 426–433. Sheng, G.P., Yu, H.Q., Li, X.Y., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894. Simon, J., Hederstedt, L., 2011. Composition and function of cytochrome c biogenesis System II. FEBS J. 278, 4179–4188. Sliekers, A.O., Derwort, N., Gomez, J.C., Strous, M., Kuenen, J., Jetten, M., 2002. Completely autotrophic nitrogen removal over nitrite in one single reactor. Water Res. 36, 2475–2482. Strous, M., Heijnen, J., Kuenen, J.G., Jetten, M., 1998. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589–596. Sun, Y., Guan, Y., Wang, H., Wu, G., 2019. Autotrophic nitrogen removal in combined nitritation and Anammox systems through intermittent aeration and possible microbial interactions by quorum sensing analysis. Bioresour. Technol. 272, 146–155. Tan, L., Chan, M.K., Saddler, J., 1984. A modification of the Lowry method for detecting protein in media containing lignocellulosic substrates. Biotechnol. Lett. 6, 199–204. Tang, X., Guo, Y., Jiang, B., Liu, S., 2018a. Metagenomic approaches to understanding bacterial communication during the anammox reactor start-up. Water Res. 136, 95–103. Tang, X., Guo, Y., Wu, S., Chen, L., Tao, H., Liu, S., 2018b. Metabolomics uncovers the
8
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Response of nitrogen removal performance, functional genes abundances and N-acyl-homoserine lactones release to carminic acid of anammox biomass
T
⁎
Lingjie Liu, Min Ji, Fen Wang , Zhao Yan, Zhongke Tian School of Environmental Science and Engineering, Tianjin University, Tianjin 300350, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Carminic acid Anammox N-acyl-homoserine lactones Quantitative reverse transcription polymerase chain reaction
Carminic acid (CA) can serve as a redox mediator and influence the electron transfer process. CA dosages of 0–5 mg/L were added to anaerobic ammonia oxidation (anammox) biomass. The results illustrated that CA not only reduced the inorganic nitrogen removal efficiency, but also decreased the nitrogen removal rate. The deterioration of nitrogen removal performance was due to the excess production of nitrate-nitrogen. The concentration of extracellular polymeric substances showed a decrease together with a decline in N-acyl-homoserine lactones release. CA addition decreased the activity of anammox bacteria while increasing the nitrifying potential. Quantitative reverse transcription polymerase chain reaction showed a decrease in anammox functional genes (nirS, hzo, and hzsB) and promotion of the expression of the nxrB gene, which corresponded with a decrease in anammox bacteria activity and the improvement of nitrifying potential. As a result, CA should not be added to anammox biomass.
1. Introduction The anaerobic ammonia oxidation (anammox) process is a biological nitrogen removal process by which ammonium-nitrogen (NH+4 - N ) is directly transferred to nitrogen gas using nitrite-nitrogen (NO−2 - N ) as
⁎
the electron acceptor (Jetten et al., 2001). Compared with traditional biological nitrogen removal technologies (Klein et al., 2017a; Klein et al., 2017b), anammox is recognized as a sustainable and environmentally friendly process for inorganic nitrogen removal in wastewater treatment because the process does not require oxygen or
Corresponding author at: Peiyang Park Campus: No. 135 Yaguan Road, Haihe Education Park, 300350 Tianjin, China. E-mail address: [email protected] (F. Wang).
https://doi.org/10.1016/j.biortech.2019.122567 Received 30 September 2019; Received in revised form 2 December 2019; Accepted 4 December 2019 Available online 07 December 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
2. Materials and methods
organic carbon and reduces the carbon dioxide and nitrous oxide emissions (Lackner et al., 2014; Rikmann et al., 2018; Zekker et al., 2019). This process can reduce the cost of wastewater treatment (Rikmann et al., 2018; Zekker et al., 2019). A redox mediator is a molecule that can function as an electron shuttle between electron acceptors and bacteria (Rabaey et al., 2005). Electron shuttles have the ability of transferring electrons between an extensive variety of compounds in redox reactions, including inorganic and organic compounds (Van der Zee and Cervantes, 2009). Various redox mediators have been applied in wastewater treatment, especially the microbial electron transfer process (Van der Zee and Cervantes, 2009). Qiao et al. (2014) investigated the short-term effect of three redox mediators (anthraquinone-2,6-disulfonate, 2-hydroxy-1,4-naphthoquinone, and anthraquinone-2-carboxylic acid) on the anammox process, and the results showed that the increase in the dosage of redox mediators had a more negative impact on nitrogen removal. Rikmann et al. (2014) also reported that anthraquinone-2,6-disulfonate improved the production of hydrazine (N2H4) and that the total nitrogen removal rate was further increased by 20%. Graphene oxide had been reported as an electron shuttle to increase the redox conversion process (Colunga et al., 2015). In the study conducted by Wang et al. (2013), a maximum increase of 10.26% in anammox activity together with greater extracellular polymeric substances (EPS) production was obtained with 0.1 g/L of graphene oxide. Furthermore, reduced graphene oxide was reported to have a greater ability of electron transfer than graphene oxide by approximately three orders of magnitude, with the total nitrogen removal rate and enzyme activity increasing by 10.2% and 1.5–2 fold, respectively (Wang et al., 2013). Quorum sensing (QS) refers to the cell-cell communication process of the ability of bacteria to sense their density by exchanging communication molecules called autoinducers (Jemielita et al., 2018). Recently, the metabolic pathways and communication mechanism of Nacyl-homoserine lactones (AHLs)-mediated QS regulation have been mentioned in the anammox consortia (Tang et al., 2018a; Tang et al., 2018b). In particular, N-(3-oxohexanoyl)-DL-homoserine lactone (3oxo-C6-HSL), N-hexanoyl-DL-homoserine lactone (C6-HSL), and N-octanoyl-DL-homoserine lactone (C8-HSL) regulate the electron transport carriers that influence bacterial activity (Tang et al., 2018b). Adding Ndodecanoyl-DL-homoserine lactone (C12-HSL) into the reactors can reduce the anammox process start-up period to 66 d compared with the control group start-up period of 80 d, thereby indicating that C12-HSL has the ability to promote the specific anammox activity (Zhao et al., 2018). Carminic acid (CA) is a deep red anthraquinone that is extracted from scale insects, and it is widely used as a pigment in different fields, e.g., crimson ink, paints, cosmetics, and food coloring (Dapson, 2007). CA has the redox group of anthraquinone and the oxidation group of hydroquinone, and it can act as a redox mediator (Li et al., 2013). Wolf et al. (2009) explored the effects of quinones containing various redox functional groups on microbial ferrihydrite reduction kinetics by Geobacter metallireducens at low concentrations, and found that CA concentrations of 0.1–10.0 μm had no apparent effect on the iron reduction kinetics. Li et al. (2013) also investigated the effects of CA on goethite reduction and current production by Klebsiella pneumonia L17, and the result showed that there was no clear effect on Fe(III) reduction. To date, study on CA addition to anammox biomass was lacking. In this study, different concentrations of exogenous CA were added to anammox biomass, and the following aspects were determined: (1) the response of nitrogen removal performance, (2) the change in QS signal molecule concentrations and EPS concentrations, and (3) the variation of anammox bacteria activity, nitrifying potential, and the change in functional genes expression activities according to quantitative reverse transcription polymerase chain reaction (qRT-PCR) after CA addition.
2.1. Original anammox biomass and CA The anammox biomass was obtained from a 4 L biofilm reactor that had been operating in a laboratory for 1.5 years with a stable total inorganic nitrogen (TIN) removal efficiency of more than 80%, with 100 mg/L of NH+4 - N and 132 mg/L of NO−2 - N in the influent under 35 ± 1 ℃. The hydraulic retention time was maintained at 24 h. CA (CAS No. 1260-17-9) was purchased from Dr. Ehrenstorfer GmbH (Germany). 2.2. Set up of batch experiments Serum flasks with an effective volume of 250 mL were used for batch exposure assays, and the volatile suspended solids (VSS) concentration was 2200 mg/L in each flask. Before feeding into the flask, anammox biomass was washed in phosphate-buffered saline (PBS) (0.14 g/L of KH2PO4 and 0.75 g/L of K2HPO4) three times in order to eliminate the residual nitrogen in the biomass. The synthetic wastewater contained 50 mg N/L of (NH4)2SO4 and 50 mg N/L of Na2NO2 as the ammonia and nitrite sources, respectively, 500 mg/L of NaHCO3 for alkalinity addition, and 100 mg/L of MgSO4∙7H2O, 180 mg/L of CaCl2⋅2H2O, and 27 mg/L of KH2PO4. In addition, 1 mL/L of trace element solutions Ⅰ and Ⅱ were added to the influent (Sliekers et al., 2002). The liquid was purged with highly purified nitrogen gas for at least 10 min to remove dissolved oxygen (DO). Six sets of batch experiments were conducted with CA concentrations of 0, 0.05, 0.1, 0.5, 1, and 5 mg/L, and each set of experiments was conducted in triplicate. The control group was set as 0 mg/L of CA addition. The original pH was fixed at 7.8 by 1 M HCl and 1 M NaOH. The operating temperature of the shaker was maintained at 37 ± 0.2 ℃ and the shaker speed was 170 rpm to maintain full contact. Liquid samples were gathered by a syringe with a long needle and periodically purged through 0.45 μm membranes for measurement of nitrogen species. 2.3. Analytical methods The concentration of nitrate-nitrogen (NO−3 - N ) was determined by ion-exchange chromatography (IC-1100, Thermo Fisher, USA). NH+4 - N , NO−2 - N , mixed liquor suspended solids, and VSS concentrations were detected based on the APHA Standard Methods (APHA, 2005). The concentration of TIN was calculated as the sum of NH+4 - N , NO−2 - N , and NO−3 - N . DO and pH were determined by a portable DO meter (HQ 30d, HACH, USA) and a digital pH meter (JENCO Model 6010, China), respectively. 2.4. Determination of anammox bacteria activity and nitrifying potential Anammox biomass was gathered at the end of 48 h to test the anammox bacteria activity and nitrifying potential. The anammox activity test was described by Dapena-Mora et al. (2010). Briefly, biomass samples were washed with PBS solution three times, and substrates of 20 mg/L of NH+4 - N and 20 mg/L of NO−2 - N were added. Each serum bottle contained approximately 400 mg VSS/L of biomass and was stripped with nitrogen gas for 10 min. Supernatants were obtained at intervals of 30 min to measure the NH+4 - N and NO−2 - N concentrations in order to calculate the anammox bacteria activity. This test was conducted in duplicate and the average value and standard deviation were calculated for analysis. For the nitrifying potential test, 20 mg/L of NH+4 - N or NO−2 - N was fed separately with adequate aeration. The liquid sampling interval was 30 min (Rich et al., 2008; Sun et al., 2019) and the concentrations of NH+4 - N , NO−2 - N , and NO−3 - N were detected to test the nitrifying potential (Almstrand et al., 2011). 2
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
NH+4 - N and NO−2 - N concentrations at the end of the reaction. However, NO−3 - N production was simulated under different CA dosages. When the reaction finished, the NO−3 - N concentrations of different CA dosages showed an increase from 13.40 mg/L to 31.07 mg/L (Fig. 1c). The nitrogen removal loading rates were 44.5, 38.6, 38.4, 37.8, 37.1, and 34.2 mg/(L·d), respectively, with CA dosages of 0–5 mg/L. The TIN removal efficiencies showed a decrease from 79.47% to 61.13% with the increase in CA dosages from 0 mg/L to 5 mg/L, respectively. In addition, the stoichiometric coefficient of ∆NH+4 - N:∆NO−3 -N described by Strous et al. (1998) was 0.26. The values of ∆NH+4 - N:∆NO−3 -N were 0.26, 0.50, 0.51, 0.53, 0.56, and 0.66 (p less than 0.01) with CA dosages from 0 mg/L to 5 mg/L. It was clear that the generation of NO−3 - N was greater than the theoretical value after the addition of CA. The decrease in TIN removal efficiency in this study was due to the excess production of NO−3 - N . The TIN degradation in this study followed a zero-order reaction kinetic model. Parameters of the zero-order kinetic model were summarized in Table 1. The results demonstrated that the TIN removal rate of the control group of 1.96 mg N/(L·h) was higher than those of the groups with CA addition, which were 1.71 mg N/(L·h), 1.70 mg N/(L·h), 1.64 mg N/(L·h), 1.62 mg N/(L·h), and 1.44 mg N/(L·h), respectively. The results indicated that CA reduced the nitrogen removal rate. It was clear that the presence of a certain amount of CA not only increased the NO−3 - N production, which resulted in the reduction in TIN removal efficiency, but also decreased the inorganic nitrogen reduction rate.
2.5. Extracellular polymeric substances extraction and analysis Biomass samples were gathered after the experiment was finished (48 h) for EPS analysis. A modified method with heat extraction was utilized for EPS extraction, and polysaccharides (PS) and proteins (PN) were determined by the Lowry-Folin method (Tan et al., 1984) using bovine serum albumin (Sigma-Aldrich) as the standard and by the phenol–sulfuric acid method using glucose (Yuanli Co., Ltd., Tianjin, China) as the standard (Nielsen, 2010), respectively. 2.6. AHLs extraction and measurement Standard AHLs (> 97%, HPLC), including C8-HSL, C12-HSL, and N(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-HSL), were purchased from Sigma-Aldrich. The AHLs extraction procedures from the water and biomass phases were described by Sun et al. (2019), and some modification was made. Briefly, at the end of the exposure experiment, 100 mL of supernatant was filtered through 0.45 μm membranes and concentrated by solidphase extraction. After drying for 30 min and elution with 5 mL of acetonitrile, liquid samples were evaporated to dryness with nitrogen gas and further re-dissolved in 0.1 mL of acetonitrile for UPLC-MS/MS (Waters, USA) analysis. As for the AHLs extraction in biomass, 25 mL sludge samples were collected and centrifuged for 5 min at 4000 rpm to remove the supernatant. After freeze-drying for 36 h at −80 ℃, 10 mL of ethyl acetate was added to extract the AHLs by ultrasonic extraction (400 W, 15 min; KQ-300DE, Kunshan, China) and a vortex process. This pretreatment was repeated three times. The mixers were conducted at 4000 rpm for 15 min and supernatant was gathered. Further steps were the same as those for the water phase mentioned above.
3.2. Effect of CA addition on anammox bacteria activity and nitrifying potential The microbial activity of anammox bacteria and the nitrifying potential under different CA dosages were illustrated in Fig. 2a and b, respectively. The activity of anammox bacteria showed a decrease with CA addition from 0 mg/L to 5 mg/L. The anammox bacteria activity in the control group was 3.73 mg N/g VSS, which was 1.96 times higher than that of the group with 5 mg/L of CA addition. The exogenous CA dosages of 0.05 mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L and 5 mg/L decreased the anammox bacteria activities by 26%, 34%, 41%, 44%, and 49%, respectively. As for the nitrifying potential, the removal rates of NH+4 - N and − NO2 - N were approximately 0.20 mg N/g VSS and 2.00 mg N/g VSS, respectively, with 0.1 mg/L of CA addition, while they increased to 0.48 mg N/g VSS and 2.78 mg N/g VSS, respectively, with a CA dosage of 5 mg/L. In contrast with the anammox bacteria activity, the nitrifying potential showed an increasing trend with the increase in CA addition. As reported in a previous study, some exogenous quinones would cause cell toxicity because of toxic organic compounds (Bolton and Dunlap, 2016). In addition, the activities of enzymes, such as nitrite reductase and hydrazine dehydrogenase, are inhibited with the addition of more than 0.6 mM 2-hydroxy-1,4-naphthoquinone (Qiao et al., 2014). Ahn (2006) observed a significant correlation between the nitrogen removal rate and microbial activity. Previous studies indicated that the nitrogen removal rate or nitrogen removal efficiency decreased in the anammox process together with the reduction in anammox activity (Jin et al., 2013). In this study, the decrease in anammox bacteria activity was accompanied by a downward trend in the NO+4 - N and NO−2 - N removal rates. The increased concentrations of NO−3 - N at the end of the experiment were attributed to the enhancement of the nitrifying potential, which corresponded with the study conducted by Poot et al. (2016). This led to a decrease in TIN removal efficiency.
2.7. RNA isolation, reverse transcription, and qRT-PCR analysis Biomass samples were collected at the end of 48 h. After the samples were centrifuged at 10 000 rpm for 5 min, a Direct-zol RNA MiniPrep kit was used for RNA extraction (Zymo Research, CA). The RNA concentrations were measured using a Nanodrop spectrophotometer (Quawell UV–vis spectrophotometer Q5000, USA). In addition, RNA was used for cDNA synthesis by reversing the transcription process using the HiScript Ⅱ 1st Strand cDNA Synthesis Kit (Vazyme). The detailed process of establishing clone libraries is presented in the Supplementary Data. qRT-PCR was conducted by an iQ5 Bio-Rad thermal cycler adopting SYBR green chemistry. Each reaction mixture included 2 × TB Green Premix EX Taq Ⅱ (Takara) (12.5 μL), forward primer (10 mM, 1 μL), reverse primer (10 mM, 1 μL), template cDNA (1 μL) and sterile deionized water (9.5 μL). The PCR program was initiated with denaturation for 2 min at 95 ℃, followed by 40 cycles of 5 s at 95 ℃, 60 s at the annealing temperature, and 30 s at 72 ℃. The primers for qRT-PCR are summarized in the Supplementary Data. Triplicate data assays were conducted using properly diluted samples and negative controls (Bustin and Nolan, 2004). In addition, the relative abundance of functional gene expression was analyzed using the 2−ΔΔCt method, and error bars represented the standard errors (Lin et al., 2016). 3. Results and discussion 3.1. Response of nitrogen removal performance to CA addition Fig. 1 illustrated the short-term response of nitrogen removal performance to CA addition in the anammox system. During the batch tests, the nitrogen removal performance of experimental groups worsened compared with that of the control group. After 48 h of exposure, the concentrations of NH+4 - N and NO−2 - N were maintained at approximately 10 mg/L and 0 mg/L, respectively (Fig. 1a and b). CA presented in the anammox system had no significant impact on the
3.3. Variation of the amount and composition of EPS EPS, which are mainly secreted from microbial organisms, produced by cellular lysis, and hydrolyzed by macromolecules, are distributed both outside cells and inside microbial aggregates (Sheng et al., 2010). 3
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
Fig. 1. NH+4 - N (a), NO−2 - N (b), NO− 3 - N (c) concentrations and the TIN removal efficiencies (d) under different CA doses with influent TIN loading rate of 55.95 mg/ (L·d).
the decrease in EPS concentration was associated with the increase in CA concentration. The EPS concentration decreased by 5.88% at 0.05 mg/L of CA addition, and further decreased by 31.46% at 5 mg/L of CA addition. Previous studies demonstrated that more EPS secretion had a positive effect on nitrogen removal performance (Miao et al., 2018). Zhang et al. (2017) thought that the reduction in PN from 271.5 mg/g VSS to 218.7 mg/g VSS might result from the suppressed metabolic activity of anammox under metallic nanoparticle stress. Similarly, the reduction in EPS occurred with the decrease in TIN removal efficiency in this study. The ratio of PN/PS reflected the surface hydrophobicity of the sludge (Yan et al., 2015). When the ratio increased, the surface properties of the sludge could effectively change, thereby contributing to the cohesion between the aggregates and the tightness and stability of the structure (McSwain et al., 2005). The ratios of PN/PS with CA dosages
Table 1 Kinetic parameters of TIN removal under different CA doses. CA doses (mg/L)
K0 (mg N/(L·h))
R2
0 0.05 0.1 0.5 1 5
1.96 1.71 1.70 1.64 1.62 1.44
0.9815 0.9820 0.9774 0.9778 0.9594 0.9401
PN and PS are the dominant components of EPS (Sheng et al., 2010). As shown in Fig. 3a, the concentrations of EPS were 77.21 mg/g VSS, 72.00 mg/g VSS, 63.71 mg/g VSS, 60.20 mg/g VSS, 56.18 mg/g VSS, and 52.43 mg/g VSS under different CA dosages. It could be seen that
Fig. 2. Variation of anammox bacteria activities (a), nitrifying potential (b) under different CA doses. 4
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
Fig. 3. EPS concentrations (a) and PN/PS (b) under different CA addition.
from 0 mg/L to 5 mg/L were 3.85, 2.43, 2.07, 2.25, 2.09, and 2.00. There was a decrease in PN/PS as the CA concentration increased in the anammox system. The addition of CA in the anammox system might have caused the deterioration of the sludge stability. 3.4. Variation of AHLs release under CA addition QS can orchestrate gene expression programs among bacteria and further underlie collective activities (Papenfort and Bassler, 2016). It has been proven that Candidatus Kuenenia, a prevailing genus in the anammox process (He et al., 2018), contains hdtS, which is a type of signal molecule synthesis gene, and has the potential to synthesize AHLs (Sun et al., 2019). C8-HSL, C12-HSL, and 3-oxo-C8-HSL were detected in both the water and biomass phases in this experiment (Fig. 4). The dominant QS signal molecules were C8-HSL and C12-HSL in the water and biomass phases, respectively. There was an apparent decrease in the C8-HSL and C12-HSL concentrations in the water phase from 6.93 ng/L to 2.48 ng/L and from 3.45 ng/L to 0.38 ng/L, respectively, with CA addition from 0 mg/L to 5 mg/L. The concentration of 3-oxo-C8-HSL was maintained at approximately 0.88 ng/L when the CA addition was less than 0.5 mg/ L, and then showed a slight decrease to 0.54 ng/L with a CA dosage of 5 mg/L. Similarly, the QS signals in the biomass also demonstrated an overall decreasing trend. The concentrations of C12-HSL were 46.98 ng/g VSS, 22.43 ng/g VSS, 23.91 ng/g VSS, 15.26 ng/g VSS, 14.56 ng/g VSS, and 7.36 ng/g VSS with CA addition from 0 mg/L to 5 mg/L. The C8-HSL concentration was reduced by 40.3% with 0.05 mg/L of CA addition and then decreased by 67.8% with 5 mg/L of CA addition. The concentration of 3-oxo-C8-HSL also showed a slight decrease from 21.68 ng/L to 15.55 ng/L with CA dosages from 0 mg/L to 5 mg/L, respectively. Thus, CA had a negative effect on QS in the anammox system, especially for C8-HSL and C12-HSL in both the water and biomass phases. It had been reported by Li et al. (2014) that the adsorption peaks of CA by the FTIR spectrum were at 3366 cm−1 and 1719 cm−1, which illustrated OeH and C]O in the carboxylic group, respectively. In the study conducted by Wolf et al. (2009), it was noted that CA had no activity or only minor activity of electron shuttling in theory because of the low redox potential. In addition, previous research considered that the redox potential of CA (-500 mV) was lower than −400 mV (the lowest value of c-cytochrome), which might explain its ineffective ability of acting as an exogenous electron shuttle for microbial Fe(III) reduction (Li et al., 2013). In addition, chronoamperometry technology demonstrated that the electron accepting capacity and donating capacity of CA were relatively low, and the ability of electron transfer of CA was weak (Li et al., 2013). In this study, CA addition reduced the rate of electron transfer and further weakened the communication among bacteria, thereby resulting in the decrease in AHLs release.
Fig. 4. The variation of AHLs in water (a) and biomass (b) phases under different CA addition.
The EPS concentrations appeared to decrease by 32.09% in this study corresponding with the decrease in QS signal concentrations. A similar result from Zhao et al. (2016) showed that porcine kidney acylase Ⅰ as well as vanillin could inactivate AHLs and lead to anammox activity reduction at an enzyme level. It had been reported that C6-HSL and C8-HSL were overproduced, especially when nitrite-oxidizing bacteria (NOB) activity was promoted (Mellbye et al., 2017). However, the C6-HSL and C8-HSL concentrations showed a decreasing trend while NO−3 - N showed an increasing trend in this study, and the specific reasons underlying this phenomenon needed to be confirmed.
5
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
Fig. 5. The effects of different doses of CA on the activity of functional genes by qRT-PCR, nirS (a), hzsB (b), hzo (c), nxrB (d) and ccsB (e).
showed a decline, and were approximately 0.24 fold and 0.05 fold greater than that of the control, respectively, with CA addition of 5 mg/ L. This result indicated that CA might restrain the anammox process in N2H4 generation. Furthermore, as reported previously, AHLs influenced the expression of hzsA, which regulated the anammox activity (Tang et al., 2019). Thus, the decreasing trend of AHLs concentrations regulated the anammox genes expression and controlled the activity of anammox to some extent. The nxrB gene is considered a phylogenetic marker and functional gene for Nitrospira, which could oxide NO−2 - N to NO−3 - N . Unlike the trend of anammox functional genes, the level of nxrB gene expression showed a slight increase from 4.96 fold to 9.78 fold greater than the control with 0.01–5 mg/L of CA addition. As described in Section 3.1, the concentration of NO−3 - N at the end of the batch experiments
3.5. Variation of the activity of functional genes by qRT-PCR under CA addition qRT-PCR technology is widely used in gene expression studies (Hu et al., 2016). To study the influence of CA addition on the anammox biomass at the transcriptional level, an expression assay of genes encoding NIR, HZS, HZO, NXR, and CCS was performed. The relative levels of different functional genes were normalized to the control group with a value of 1, and were shown in Fig. 5. The nirS gene, which is catalyzed by nitric oxide oxidoreductase (NirS), can reduce NO−2 - N to NO, which is the first step in anammox reaction. The expression of the nirS gene showed a slight decrease and was finally maintained at around 0.08 fold greater than that of the control group, which corresponded with the decrease in the NO−2 - N reduction rate. The expression of anammox-related functional genes, namely hzsB and hzo, also
6
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
References
increased to 31.07 mg/L, which was the main reason for the decrease in the TIN removal efficiency and corresponded with the higher level of nxrB gene expression. The chemical properties offered by heme are utilized in multiple PN that implement an array of functions, e.g., redox catalysis and electron transfer (Poulos, 2014). Heme c is covalently bound, and has the ability of catalysis of various redox reactions as well as electron transfer (Bertini et al., 2006). ccsB is one of the components of the cytochrome c synthase (Simon and Hederstedt, 2011). After CA addition from 0.01 mg/L to 5 mg/L, the expression of the ccsB gene showed a decreasing trend from 0.71 to 0.13 fold compared with control group, which indicated that enzyme synthesis of cytochrome c was repressed. Furthermore, the electron transport and communication among bacteria were reduced, which caused the deterioration of nitrogen removal performance. Overall, the qRT-PCR results revealed that the expression of anammox-related genes weakened and that of nitrification-related genes strengthened, which was in accordance with the variation of anammox activity and nitrifying potential. As a result, it led to the excess production of NO−3 - N and decreased TIN removal efficiency. The maintenance of anammox bacteria and suppression of NOB were the key issues for maintaining operational stability of the anammox process (Persson et al., 2014). It had been reported that the multiplication of NOB resulted in irreversible inhibition of the anammox system because of competition with NO−2 - N as an electron acceptor (Joss et al., 2011). In summary, in order to maintain the stability of the anammox system, CA should not be added to the system.
Ahn, Y.H., 2006. Sustainable nitrogen elimination biotechnologies: a review. Process. Biochem. 41, 1709–1721. Almstrand, R., Lydmark, P., Sörensson, F., Hermansson, M., 2011. Nitrification potential and population dynamics of nitrifying bacterial biofilms in response to controlled shifts of ammonium concentrations in wastewater trickling filters. Bioresour. Technol. 102, 7685–7691. APHA, A., WEF, 2005. Standard methods for the examination of water and wastewater. 21 st ed. Washington, DC. Bertini, I., Cavallaro, G., Rosato, A., 2006. Cytochrome c: occurrence and functions. Chem. Rev. 106, 90–115. Bolton, J.L., Dunlap, T., 2016. Formation and biological targets of quinones: cytotoxic versus cytoprotective effects. Chem. Res. Toxicol. 30, 13–37. Bustin, S.A., Nolan, T., 2004. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Technol. 15, 155. Colunga, A., Rangel-Mendez, J.R., Celis, L.B., Cervantes, F.J., 2015. Graphene oxide as electron shuttle for increased redox conversion of contaminants under methanogenic and sulfate-reducing conditions. Bioresour. Technol. 175, 309–314. Dapena-Mora, A., Vázquez-Padín, J., Campos, J., Mosquera-Corral, A., Jetten, M., Méndez, R., 2010. Monitoring the stability of an Anammox reactor under high salinity conditions. Biochem. Eng. J. 51, 167–171. Dapson, R., 2007. The history, chemistry and modes of action of carmine and related dyes. Biotech. Histochem. 82, 173–187. He, S., Chen, Y., Qin, M., Mao, Z., Yuan, L., Niu, Q., Tan, X., 2018. Effects of temperature on anammox performance and community structure. Bioresour. Technol. 260, 186–195. Hu, Y., Deng, T., Chen, L., Wu, H., Zhang, S., 2016. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata. PLoS One 11, e0154384. Jemielita, M., Yan, J., Hurley, A., Stone, H., Wingreen, N., Bassler, B., 2018. Quorum Sensing Control of Vibrio Choleraeaggregation. APS Meeting Abstracts. Jetten, M.S., Wagner, M., Fuerst, J., van Loosdrecht, M., Kuenen, G., Strous, M., 2001. Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Curr. Opin. Biotechnol. 12, 283–288. Jin, R.C., Ma, C., Yu, J.J., 2013. Performance of an Anammox UASB reactor at high load and low ambient temperature. Chem. Eng. J. 232, 17–25. Joss, A., Derlon, N., Cyprien, C., Burger, S., Szivak, I., Traber, J., Siegrist, H., Morgenroth, E., 2011. Combined nitritation–anammox: advances in understanding process stability. Environ. Sci. Technol. 45, 9735–9742. Klein, K., Kattel, E., Goi, A., Kivi, A., Dulova, N., Saluste, A., Zekker, I., Trapido, M., Tenno, T., 2017a. Combined treatment of pyrogenic wastewater from oil shale retorting. Oil Shale 34, 82–96. Klein, K., Kivi, A., Dulova, N., Zekker, I., Mölder, E., Tenno, T., Trapido, M., Tenno, T., 2017b. A pilot study of three-stage biological–chemical treatment of landfill leachate applying continuous ferric sludge reuse in Fenton-like process. Clean Technol. Environ. Policy 19, 541–551. Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H., van Loosdrecht, M.C., 2014. Full-scale partial nitritation/anammox experiences–an application survey. Water Res. 55, 292–303. Li, B., Li, Z., Situ, B., Dai, Z., Liu, Q., Wang, Q., Gu, D., Zheng, L., 2014. Sensitive HIV-1 detection in a homogeneous solution based on an electrochemical molecular beacon coupled with a nafion–graphene composite film modified screen-printed carbon electrode. Biosens. Bioelectron. 52, 330–336. Li, X., Liu, L., Liu, T., Yuan, T., Zhang, W., Li, F., Zhou, S., Li, Y., 2013. Electron transfer capacity dependence of quinone-mediated Fe (III) reduction and current generation by Klebsiella pneumoniae L17. Chemosphere 92, 218–224. Lin, W.P., Xiong, G.P., Lin, Q., Chen, X.W., Zhang, L.Q., Shi, J.X., Ke, Q.F., Lin, J.H., 2016. Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury. J. Neuroinflamm. 13, 52. McSwain, B., Irvine, R., Hausner, M., Wilderer, P., 2005. Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Appl. Environ. Microbiol. 71, 1051–1057. Mellbye, B.L., Spieck, E., Bottomley, P.J., Sayavedra-Soto, L.A., 2017. Acyl-homoserine lactone production in nitrifying bacteria of the genera Nitrosospira, Nitrobacter, and Nitrospira identified via a survey of putative quorum-sensing genes. Appl. Environ. Microbiol. 83, e01540–17. Miao, L., Zhang, Q., Wang, S., Li, B., Wang, Z., Zhang, S., Zhang, M., Peng, Y., 2018. Characterization of EPS compositions and microbial community in an Anammox SBBR system treating landfill leachate. Bioresour. Technol. 249, 108–116. Nielsen, S.S., 2010. Phenol-sulfuric acid method for total carbohydrates. In: Heifelberg, D. (Ed.), Food Analysis Laboratory Manual, Second ed. Springer, New York, pp. 47–55. Papenfort, K., Bassler, B.L., 2016. Quorum sensing signal–response systems in Gram-negative bacteria. Nat. Rev. Microbiol. 14, 576. Persson, F., Sultana, R., Suarez, M., Hermansson, M., Plaza, E., Wilén, B.-M., 2014. Structure and composition of biofilm communities in a moving bed biofilm reactor for nitritation–anammox at low temperatures. Bioresour. Technol. 154, 267–273. Poot, V., Hoekstra, M., Geleijnse, M.A., van Loosdrecht, M.C., Pérez, J., 2016. Effects of the residual ammonium concentration on NOB repression during partial nitritation with granular sludge. Water Res. 106, 518–530. Poulos, T.L., 2014. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962. Qiao, S., Tian, T., Zhou, J., 2014. Effects of quinoid redox mediators on the activity of anammox biomass. Bioresour. Technol. 152, 116–123. Rabaey, K., Boon, N., Höfte, M., Verstraete, W., 2005. Microbial phenazine production enhances electron transfer in biofuel cells. Environ. Sci. Technol. 39, 3401–3408. Rich, J.J., Dale, O.R., Song, B., Ward, B.B., 2008. Anaerobic ammonium oxidation
4. Conclusion EPS appeared to decrease after CA addition to the anammox system. The AHLs concentrations also decreased, especially C8-HSL in the water phase and C12-HSL in the biomass phase. CA inhibited anammox bacteria activity by 49% and promoted the nitrifying potential to 2.78 mg N/g VSS. The qRT-PCR results indicated that the expression of anammox-related genes (nirS, hzsB, and hzo) was weakened and that of nitrification-related genes (nxrB) was strengthened. This led to the excess production of NO−3 - N and decrease TIN removal efficiency. Overall, CA addition should be avoided to maintain the stability of the anammox system. CRediT authorship contribution statement Lingjie Liu: Writing - original draft, Conceptualization, Methodology, Investigation, Data curation, Visualization, Formal analysis. Min Ji: Supervision. Fen Wang: Writing - review & editing, Supervision, Funding acquisition. Zhao Yan: Methodology. Zhongke Tian: Methodology. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment of China (2017ZX07106). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122567. 7
Bioresource Technology 299 (2020) 122567
L. Liu, et al.
regulatory pathway of acyl-homoserine lactones based quorum sensing in anammox consortia. Environ. Sci. Technol. 52, 2206–2216. Tang, X., Guo, Y., Zhu, T., Tao, H., Liu, S., 2019. Identification of quorum sensing signal AHLs synthases in Candidatus Jettenia caeni and their roles in anammox activity. Chemosphere 225, 608–617. Van der Zee, F.P., Cervantes, F.J., 2009. Impact and application of electron shuttles on the redox (bio) transformation of contaminants: a review. Biotechnol. Adv. 27, 256–277. Wang, D., Wang, G., Zhang, G., Xu, X., Yang, F., 2013. Using graphene oxide to enhance the activity of anammox bacteria for nitrogen removal. Bioresour. Technol. 131, 527–530. Wolf, M., Kappler, A., Jiang, J., Meckenstock, R.U., 2009. Effects of humic substances and quinones at low concentrations on ferrihydrite reduction by Geobacter metallireducens. Environ. Sci. Technol. 43, 5679–5685. Yan, L., Liu, Y., Wen, Y., Ren, Y., Hao, G., Zhang, Y., 2015. Role and significance of extracellular polymeric substances from granular sludge for simultaneous removal of organic matter and ammonia nitrogen. Bioresour. Technol. 179, 460–466. Zekker, I., Kivirüüt, A., Rikmann, E., Mandel, A., Jaagura, M., Tenno, T., Artemchuk, O., Rubin, S., Tenno, T., 2019. Enhanced efficiency of nitritating-anammox sequencing batch reactor achieved at low decrease rates of oxidation–reduction potential. Environ. Eng. Sci. 36, 350–360. Zhang, Z.Z., Xu, J.J., Shi, Z.J., Cheng, Y.F., Ji, Z.Q., Deng, R., Jin, R.C., 2017. Short-term impacts of Cu, CuO, ZnO and Ag nanoparticles (NPs) on anammox sludge: CuNPs make a difference. Bioresour. Technol. 235, 281–291. Zhao, R., Zhang, H., Zhang, F., Yang, F., 2018. Fast start-up anammox process using Acylhomoserine lactones (AHLs) containing supernatant. J. Environ. Sci. 65, 127–132. Zhao, R., Zhang, H., Zou, X., Yang, F., 2016. Effects of Inhibiting Acylated Homoserine Lactones (AHLs) on Anammox Activity and Stability of Granules’. Curr. Microbiol. 73, 108–114.
(anammox) in Chesapeake Bay sediments. Microb. Ecol. 55, 311–320. Rikmann, E., Zekker, I., Tenno, T., Saluste, A., Tenno, T., 2018. Inoculum -free start-up of biofilm- and sludge-based deammonification systems in pilot scale. Int. J. Environ. Sci. Technol. 15, 133–148. Rikmann, E., Zekker, I., Tomingas, M., Vabamäe, P., Kroon, K., Saluste, A., Tenno, T., Menert, A., Loorits, L., dC Rubin, S.S., 2014. Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors. J. Biosci. Bioeng. 118, 426–433. Sheng, G.P., Yu, H.Q., Li, X.Y., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894. Simon, J., Hederstedt, L., 2011. Composition and function of cytochrome c biogenesis System II. FEBS J. 278, 4179–4188. Sliekers, A.O., Derwort, N., Gomez, J.C., Strous, M., Kuenen, J., Jetten, M., 2002. Completely autotrophic nitrogen removal over nitrite in one single reactor. Water Res. 36, 2475–2482. Strous, M., Heijnen, J., Kuenen, J.G., Jetten, M., 1998. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589–596. Sun, Y., Guan, Y., Wang, H., Wu, G., 2019. Autotrophic nitrogen removal in combined nitritation and Anammox systems through intermittent aeration and possible microbial interactions by quorum sensing analysis. Bioresour. Technol. 272, 146–155. Tan, L., Chan, M.K., Saddler, J., 1984. A modification of the Lowry method for detecting protein in media containing lignocellulosic substrates. Biotechnol. Lett. 6, 199–204. Tang, X., Guo, Y., Jiang, B., Liu, S., 2018a. Metagenomic approaches to understanding bacterial communication during the anammox reactor start-up. Water Res. 136, 95–103. Tang, X., Guo, Y., Wu, S., Chen, L., Tao, H., Liu, S., 2018b. Metabolomics uncovers the
8