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A fast start-up of the organotrophic anammox process inoculated with constructed wetland sediment
Ecological Engineering 138 (2019) 454–460
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A fast start-up of the organotrophic anammox process inoculated with constructed wetland sediment
T
Xuejiao Yina, Jun Zhaia, , Wei Hua, Yue Lia, Md. Hasibur Rahamana,c, Jacek Mąkiniab ⁎
a
Chongqing University, Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Chongqing 400045, China Department of Sanitary Engineering, Gdańsk University of Technology, 80-233 Gdansk, Poland c Department of Environmental Science and Technology, Jessore University of Science and Technology, Jessore, 7408, Bangladesh b
ARTICLE INFO
ABSTRACT
Keywords: Anammox Organotrophic bacteria TOC/TN ratio Chloramphenicol Wetland sediment qPCR
Organotrophic anaerobic ammonium oxidation (anammox) bacteria can utilize small volatile fatty acids with nitrate as electron acceptors with less energy consumption and no biomass production. To achieve a faster and stable start-up of organotrophic anammox process, in this study, the growth of organotrophic anammox bacteria seeded from hybrid constructed wetland (CW) sediment under different TOC/TN ratios and different chloramphenicol concentrations were investigated. The incubation study was conducted at the TOC/TN ratio = 0.0375–0.1 or 0.1–0.2 for the period of over five months by using serum bottles. The anammox bacteria revealed a higher activity when the TOC/TN ratio was 0.1, with the removal efficiency of NH4+-N (60–80%) and NO2−-N (~100%). The relative abundances of anammox in the incubated CW sediment were about 30% higher in comparison with the municipal waste water treatment plant sludge, suggesting the CW sediment could be a viable source for the enrichment of organotrophic anammox bacteria. On the contrary, the continuous addition of 50 mg/L chloramphenicol completely inhibited the anammox activity in our study. Following the results of the batch tests, Candidatus Brocadia caroliniensis was successfully enriched with the CW sediment in an autocontrolled SBR for the period of 40 days.
1. Introduction Anaerobic ammonium oxidation (anammox), firstly reported by Mulder et al. (1995), is one of the most important processes in the nitrogen cycle in both natural and engineered systems. One of the main drawbacks for practical applications of the anammox process is the long start-up period due to a very low specific growth rate of anammox bacteria, e.g. 0.014–0.09 d−1 (Zhang et al., 2017; Wang et al., 2017b; Cao et al., 2017). Activated sludge of municipal waste water treatment plant (WWTP) has commonly been used as the inoculum source, but there are studies indicating that also aerobic granular sludge, anaerobic granular sludge, and river or lake sediment could be used for the enrichment of anammox bacteria (Law et al., 2017; Wang et al., 2012). The choice of appropriate seeding sludge may be crucial for the effective enrichment of anammox bacteria and shortening the start-up period. For many years, anammox bacteria have been thought to be obligate autotrophic microorganisms which convert ammonia to nitrogen gas with nitrite as electron acceptor, coupled with the production of nitrate (Winkler et al., 2012; Xu et al., 2019). However, results of several
⁎
recent studies suggest that certain anammox species are capable of oxidizing short-chain volatile fatty acids (VFA), such as formate, acetate, and propionate, via the dissimilatory nitrate reduction pathway (without biomass production), followed by the normal anammox process (Kartal and de Almeida, 2013; Kumar and Lin, 2010; Winkler et al., 2012). The organotrophic nature of anammox would be advantageous due to consumption of nitrate and decreased production of surplus sludge. However, the actual ability of these organotrophic anammox species to oxidize VFA still remains unclear. In general, an adequate total organic carbon to total nitrogen (TOC/TN) ratio is potentially a key factor for their enrichment. Huang et al. (2014) observed that Candidatus Jettenia asiatica were capable of growing at the TOC/TN ratio of 0.1 (adjusted by sodium acetate) and 0.22 (adjusted by sodium propionate). With the addition of acetate, the abundance of Candidatus Jettenia, Candidatus Kuenenia and Candidatus Brocadia respectively increased, decreased significantly and decreased less, suggesting that the presence of VFA may favor the growth of Candidatus Jettenia (Liang et al., 2015). In contrast, Shu et al. (2016) showed that Candidatus Brocadia sinica had higher specific growth rates than Candidatus Jettenia asiatica and
Corresponding author. E-mail address: [email protected] (J. Zhai).
https://doi.org/10.1016/j.ecoleng.2019.07.039 Received 15 May 2019; Received in revised form 29 July 2019; Accepted 31 July 2019 Available online 31 August 2019 0925-8574/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 Inoculum sources, TOC/TN ratios and chloramphenicol concentrations used for the experiments. Group
Reactor
Inoculum source
TOC/TN
Chloram-phenicol (mg/L)
Phase I
Phase II
Ammonium (mg N/L)
Nitrite (mg N/L)
Phase I
Phase II
Phase I
Phase II
1
SP-1 SP-2 SP-3
SP* SP* SP*
0.0375 0.1 0.1
0.1 0.2 0.1
0 0 50
70–240 70–190 70–190
240 190 190
70–190 70–115 70–115
190 115 115
2
CW-1 CW-2 CW-3
CW* CW** CW**
0.0375 0.1 0.1
0.1 0.2 0.1
0 0 50
70–240 70–190 70–190
240 190 190
70–190 70–115 70–115
190 115 115
* Sewage treatment plant sludge (Ji Guanshi). ** Constructed wetland sediment (Bai Shiyi).
septic tank (450 m3), two parallel sedimentation ponds (200 m3 each), a lagoon (640 m3), five parallel vertical baffle flow wetlands (708 m2 total) in parallel, five parallel horizontal baffle flow wetlands (HSFWs) (2740 m2 total) in parallel, and a clean water pond (340 m3). In this study, the inoculum biomass was withdrawn from the HSFW sediments, where the COD/TN ratio was approximately 1.0 and the depth was 10 cm.
Candidatus Kuenenia stuttgartiensis in a wide range of the TOC/TN ratio (0.05 to 0.8). Furthermore, the organotrophic anammox process was found to be the main contributor to the TN loss at the TOC/TN ratio of 0.2. The anammox enrichment process may also be faster when the competitive bacteria are inhibited without negatively affecting anammox bacteria. There are studies (Koney and Morse, 2009; Maeda et al., 2017; Myrold and Posavatz, 2007; Tao and Gao, 2012) indicating that antibiotics, such as chloramphenicol (0.07 mM), amoxicillin (0.1–1.0 mM) and oxytetracycline (< 0.5 mM), can indeed inhibit the activity of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) or denitrifying heterotrophic bacteria (DNB), thus favoring the enrichment of anammox bacteria. Furthermore, chloramphenicol was proved to be helpful for the enrichment of Candidatus Brocadia anammoxidans (Toh et al., 2002). However, the effect of chloramphenicol on anammox bacteria in the presence of organic carbon has not been investigated. CWs have the advantage of occupying less area and being more economic. CWs are proper for the treatment of wastewater in towns. If organotrophic anammox process can be used in CWs, there will be an increase in the removal efficiency of nitrate. For applications of organotrophic anammox in CWs, it is necessary to investigate the start-up of organotrophic anammox in CWs. So in this study, we investigate the start-up of organotrophic anammox seeded from CW sediment. The start-up of organotrophic anammox seeded from sewage plant was also conducted as comparison. In the previous study at the Bai Shiyi CW, Zhai et al. (2016) reported the presence of Candidatus Brocadia fulgida and Candidatus anammoxoglobus propionicus, and these anammox species may indeed be capable of oxidizing VFA (Castro-Barros et al., 2017; Kartal et al., 2007). To our knowledge, there have been no studies showing that the CW sediment could be used as the anammox inoculum source. The effects of TOC/TN ratio (0.0375–0.1 vs. 0.1–0.2) and chloramphenicol (none vs. 50 mg/L) on organotrophic anammox were also investigated. The investigation of the chloramphenicol effect on anammox enrichment in the presence of organic carbon is another novel aspect of this study.
2.2. Batch reactor setup and operational conditions Six serum bottles (Shuniu, China) with the capacity of 200 ml each were used as batch reactors to carry out measurements of the capacity of nitrogen removal and investigate the optimum growth conditions of organotrophic anammox bacteria (described in point 2.3). The batch reactors were divided into two groups of three bottles in terms of the origin of the inoculum biomass. In group 1 (denoted as SP), the reactors were incubated with the activated sludge, whereas in group 2 (denoted as CW), the reactors were incubated with the CW sediment after screening on a sieve with apertures of 300 μm. The batch reactors were operated in a 24 h-cycle in a sequencing fed-batch mode. The operation stage of each cycle included four periods: about 23 h and 15 min of an anaerobic reaction in a shaker with the speed of 180 rpm and temperature of 33 °C, followed by a settling period of 20 min. Then the supernatant with half of the reactor liquid volume was withdrawn and replaced by fresh medium (about 3 min). Finally, the reactors were blown through with carbon dioxide (for 2 min) to adjust the pH to 7.4 ± 0.2 and then purged of dissolved oxygen by blowing nitrogen gas for 20 min. 2.3. TOC/TN ratios, chloramphenicol doses setting and nutrients in the fresh medium Different TOC/TN ratios (adjusted by addition of sodium acetate) and chloramphenicol doses set for the reactors were shown in Table 1. There were two operational phases for reactor SP-1, SP-2, CW-1 and CW-2. In phase I (day 0–136), termed the cultivation phase, a stable anammox reaction was achieved. In phase II (day 137–201), termed the experimental phase, factors influencing the growth of anammox bacteria were investigated. In the two phases, the ammonium and nitrite concentrations in the fresh medium were also shown in Table 1. In addition to ammonium and nitrite, the fresh medium also contained 500 mg/L NaHCO3, 300 mg/L MgSO4·7H2O, 180 mg/L CaCl2·2H2O, 56.7 mg/L KH2PO4, 1 ml/L trace elements I and 1 ml/L trace elements II. The content of both trace elements was prepared as recommended by Van de Graaf et al. (1996).
2. Material and methods 2.1. Origin of the inoculum biomass For experiments, the inoculum biomasses comprised activated sludge from Ji Guanshi SP of Chongqing (China) and sediment from Bai Shiyi CW of Chongqing (China). The Ji Guanshi SP treats approximately 1.2 × 106 m3/d of municipal wastewater using the A2/O process configuration in the biological stage. The inoculum biomass was withdrawn from the anaerobic compartment, where the COD/TN ratio was approximately 8.0. The Bai Shiyi CW is a hybrid system receiving municipal wastewater. The plant went into operation in January 2011, treating 500–600 m3/d of wastewater discharged from a community of approximately 6000 inhabitants. The hybrid CW plant consists of a
2.4. Confirmation of the optimal enrichment of organotrophic anammox bacteria In order to verify the findings obtained from the serial reactors with 455
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respect to the optimum TOC/TN and preferable inoculum, an autocontrolled sequencing batch reactor (SBR) with a working volume of 5 L was operated to enrich organotrophic anammox bacteria at the final stage of the study. The influent characterization was kept the same as in the optimum batch reactor and the cycle time was shortened to 6 h.
Statistics 20 (IBM, USA). 3. Results and discussion In reactor SP-1, CW-1, SP-2 and CW-2, both ammonium and nitrite were efficiently removed under non-aerated conditions, suggesting the presence of active anammox reaction in the four batch reactors without the addition of chloramphenicol. The behavior of ammonium, nitrite and nitrate removal capacities during the two operational phases (cultivation and experimental) in these four reactors was showed in Fig. 1. The removal efficiencies of ammonium and nitrite in these reactors can be seen in Fig. S1. However, with the addition of 50 mg/L of chloramphenicol over the period of five months, the removal of ammonium and nitrite during one reaction cycle was hardly observable in reactors SP-3 and CW-3 (Fig. S2). In our study, the continuous addition of chloramphenicol in the presence of organic carbon inhibited the activities of organotrophic anammox bacteria, as well as heterotrophic denitrifiers. Similar results were obtained during the operation of an anammox SBR and an anammox semi-CSTR with planktonic cells in presence of chloramphenicol (Ding et al., 2018; Fernandez et al., 2009). A concentration of 20 mg/L of chloramphenicol was continuously added to an anammox SBR causing a decrease of the specific anammox activity of the biomass from 0.25 to 0.05 gN (gVSS d−1). Ding et al. (2018) showed that anammox bacteria (Candidatus Kuenenia stuttgartiensis) were sensitive to sulfide (IC50 = 5uM) and chloramphenicol (IC50 = 19 mg/L). Jin et al. (2012) also reported that continuously addition of 20 mg/L of chloramphenicol would cause a decrease of 25% anammox effect. Except for chloramphenicol, Zhang et al. (2019) reported that after a long term of 2 mg/L oxytetracycline exposure, the anammox activity decreased by about 60%. Chen et al. (2017) reported that 130 mg/L thiocyanate could decrease the nitrogen removal efficiency by 26% within two days in the anammox reactor. However, some previous studies reported that a concentration of 100 mg/L of chloramphenicol was functional on repressing denitrifiers without inhibiting the activities of autotrophic anammonx bacteria (Toh et al., 2002), and the estimated IC50 value of chloramphenicol on the anammox process assessed by luminescent bacteria test was 409.9 mg/L after the exposure for 15 min (Ding et al., 2015). Moreover, Dapena-Mora et al. (2007) reported that chloramphenicol with concentrations up to 1 g/L did not inhibit the activity of Candidatus Kuenenia stuttgartiensis within the exposure for 6 h. Short periods of the exposure to chloramphenicol do not cause explicit inhibition to anammox activities. However, it is not ready to derive an unambiguous conclusion on the inhibition effect of chloramphenicol to anammox bacteria, and further, more systematic researches are still needed.
2.5. Analytical methods 10 ml samples (supernatant) were collected at the beginning and the end of each reaction cycle and filtrated by 0.45 μm filter membranes (Jin Teng, China). Then the samples were analyzed for ammonium, nitrite and nitrate concentrations. The measurements were performed using colorimetric methods by UV/VIS spectrophotometer (UV-2550, Shimadzu, Japan). The TN concentrations were estimated as a sum of those three N species. The TOC concentrations were detected by TOC analyzer (Shimadzu TOC-L machine, Japan). 2.6. Microbial analysis 2.6.1. DNA extraction and sequencing 5 ml samples were withdrawn from reactor SP-1, SP-2, CW-1 and CW-2 at the end of phase I. The microbial genome DNA was extracted from the samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the manufacturer’s protocol. The sequencing procedures were conducted as described by Wang et al. (2017a) and more details can be found in the Supporting Information (SI). The sequencing results were blasted on National Center for Biotechnology Information (NCBI). Then a consensus tree was built based on the maximum likelihood. The bar represented 5% estimated sequence divergence. All sequence descriptions followed the NCBI GenBank accession number. 2.6.2. Real-time quantitative polymerase chain reaction (qPCR) In order to evaluate the abundance of nitrogen removal related bacteria at the end of phase I, genomic DNA was extracted from 5 ml samples in the reactors and nitrogen transforming genes including anammox 16S RNA (Amox) gene, ammonia oxidation function gene (amoA) and denitrification function gene (NirS) were quantified by using qPCR. The qPCR was performed in a 15 μl reaction system containing 7.5 μl SYBR Green Premix (Genstar, China), 0.3 μl of forward and reverse primer, 0.3 μl of genomic DNA and 6.6 µl ddH2O. The primer information for qPCR was listed in Table 2. The R2 value for all the standard curve lines were higher than 0.99. The Ct value (threshold cycle) was determined to quantify the copy numbers of all studied genes, and duplexed assays were run in triplicate for each reaction. 2.6.3. Statistical analysis The absolute and relative abundances of anammox 16sRNA gene and two functional genes, namely nirS and amoA, were used in stepwise regression analyses with SPSS Statistics 20 (IBM, USA). A statistical One-Way ANOVA was also applied to analyze these results by SPSS
3.1. Achieving a stable anammox process in the cultivation phase (phase I) Within phase I, there was nearly no anammox reaction during the first two months, and the consumption of ammonium and nitrite was very unstable. At the beginning, a portion of nitrite was consumed while the concentrations of ammonium increased slightly. This implicitly occurred due to the combined effects of denitrifying bacteria, anammox bacteria and decay of bacteria that could not adapt to the new environment. As the system stabilized, the anammox reaction appeared around day 60 and became more prominent afterwards. For reactor SP-1 and CW-1, from day 117 on, the removal efficiencies of ammonium and nitrite stabilized over 63% and 96%, respectively. At the end of phase I, over 80 mg N/L of ammonium and 100 mg N/L of nitrite were removed in one reaction cycle. For reactor SP-2, the removal efficiencies of ammonium and nitrite reached 60% and 98% respectively after 89 days. At the end of phase I, approximately 60 mg N/L of ammonium and 90 mg N/L of nitrite were removed in one reaction cycle. For reactor CW-2, from day 75 on, the removal efficiencies of ammonium and nitrite reached 63% and 82%,
Table 2 Sequences, annealing temperatures and target genes of the primers used in this study. Primer
Sequence (5′–3′)
Annealing temp (°C)
Target gene
References
338F 820R
ACTCCTACGGGAGGCAGCA TTCGCAATGCCCGAAAGG
52
Sun et al. (2016)
1F 2R
GGGGTTTCTACTGGTGGT CCCCTCKGSAAAGCCTTCTTC
55
cd3AF9 R3cd
GTSAACGTSAAGGARACSGG GASTTCGGRTGSGTCTTGA
55
Anammox 16S RNA gene Ammonia oxidation gene Nitrite reductase gene
Miao et al. (2016) Coban et al. (2015)
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Fig. 1. The performances of reactor SP-1, SP-2, CW-1 and CW-2 in terms of the removal capacity of ammonium (■) nitrite ( cycle: (a) reactor SP-1; (b) reactor SP-2; (c) reactor CW-1; (d) reactor CW-2.
) and nitrate ( ) during one reaction
nitrogen removal efficiency increased with the operation time, however, when the TOC/TN ratio increased to 0.2, the nitrogen removal efficiency decreased sharply below 20%. These results suggest that too high TOC/TN ratio had a negative effect on the growth of anammox bacteria regardless of the inoculum source. There are some other reports about the effects of TOC/TN ratio on organotrophic anammox bacteria. Liang et al. (2015) showed that organotrophic anammox bacteria could be enriched by adding acetate or propionate with the TOC/TN ratio of 0.1. Shu et al. (2016) found that at the TOC/TN ratio of 0.2, organotrophic anammox made the largest contribution to nitrogen removal in the investigated range (0.05–0.82). Zhang et al. (2019a,b) reported that at the COD/TN ratio of 0.15, mixotrophic anammox consortia showed no difference on the removal of TN comparing with autotrophic anammox consortia. The difference of the value may implicitly be related to the species of anammox bacteria present in the reactors.
respectively. With the operation time going on, the removal efficiencies of ammonium and nitrite kept increasing. At day 103, the removal efficiencies reached the maximum level. Until to the end of phase I, the removal efficiencies of ammonium and nitrite stabilized over 86% and 94%, respectively. The removal load of ammonium and nitrite were approximately 83 mg N/L of and 118 mg N/L. In general, successful start-up times of the anammox process for reactor SP-1, SP-2, CW-1 and CW-2 were 117, 89, 117 and 103 days, respectively. After start-up, the ammonium and nitrite removal efficiencies kept at the maximum level. The start-up times of anammox process observed in the present study were comparable with other studies on autotrophic anammox enrichment. The start-up time, with ammonium and nitrite removal efficiencies stabilized at the maximum level in an SBR reactor, was 101 days as reported by Wang et al. (2012). Lu et al. (2018) operated three reactors using different materials as the biofilm carriers, for which the start-up times to achieve maximum removal efficiencies of nitrogen were in the range 105–145 days. Besides, it should be noticed that when the TOC/TN ratio was 0.0375, the anammox start-up times were both 117 days. While the TOC/TN ratio was 0.1, the anammox start-up times were 89, 103 days respectively. From these results, it could be concluded that organotrophic anammox process achieved faster start-up when the TOC/TN ratio was 0.1 rather than 0.0375.
3.3. Variations in the stoichiometric ratios Stoichiometric ratios can be used as indicators of the anammox process. The theoretical stoichiometric ratios of RS (consumption of nitrite to ammonium) and RP (production ratio of nitrate to consumption of the sum of ammonium and nitrite) of anammox reaction reported by Mulder et al. (1995) were 1.32 and 0.11, respectively. As shown in Fig. 3, the stoichiometric ratios of RS and RP in reactor SP-1 between 53 and 201 days were stable at about 1.44 ± 0.08:1 and 0.059 ± 0.018, respectively. Similarly, the stoichiometric ratios of RS and RP in reactor CW-1 between 75 and 201 days were about 1.24 ± 0.14:1 and 0.067 ± 0.014, respectively. The RS values of these two reactors were close to the theoretical value (1.32:1), which indicated the dominance of the anammox reaction for nitrogen removal. With the presence of organic carbon, the RP values for the two reactors were approximately 50–60% of the theoretical value (0.11).
3.2. Anammox performance after modification of the TOC/TN ratios in the experimental phase (phase II) For reactors SP-1 and CW-1 in phase I (TOC/TN = 0.0375), the nitrogen removal efficiency increased significantly with the operation time. During phase II, in which the TOC/TN ratio was increased to 0.1, the ammonium removal efficiency of reactors SP-1 and CW-1 increased by 7% and 19%, respectively, in comparison with the end of phase I (Fig. 2). When the TOC/TN ratio was 0.1 in reactor SP-2 and CW-2, the 457
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Fig. 2. The ammonium (■) and nitrite ( ) removal efficiency in the reactors at the end of phase I and phase II. a) SP group; b) CW group. Note: 0.1* the end of experimental phase (phase II) of reactor SP-1 and CW-1; 0.1** the end of cultivation phase (phase I) of reactor SP-2 and CW-2.
The portion of nitrate could implicitly be consumed by the dissimilatory nitrate reduction pathway of anammox bacteria. The stoichiometric ratios of RS and RP for reactor SP-2 were about 1.55 ± 0.11 and 0.12 ± 0.02 between 75 and 131 days (phase I), and changed to 2.28 ± 0.5 and 0.09 ± 0.04 between 145 and 201 days (phase II). The stoichiometric ratios of RS and RP for reactor CW-2 were about 1.38 ± 0.1 and 0.11 ± 0.017 between 61 and 131 days (phase I). However, the RS value for reactor CW-2 increased from 1.42 to 3.19, whereas the RP value dropped gradually from 0.029 to nearly 0.0 in phase II. For reactor SP-2 and CW-2, the RS and RP values in phase I were close to the theoretical values, indicating the occurrence of the anammox reaction. However, in phase II, the RS value increased significantly and the RP value decreased, which indicated that the anammox reaction was overpassed by denitrification for nitrogen removal.
in the reactors was constructed based on the sequencing results, as shown in Fig. 4. The sequencing analysis revealed that the anammox bacteria in reactor SP-1, CW-1, SP-2 and CW-2 had 99% similarity with Candidatus Brocadia caroliniensis. Candidatus Brocadia caroliniensis was reported to be capable of oxidizing short-chain VFAs (Park et al., 2017). The copy numbers and relative abundances of Amox, NirS and amoA genes, quantified by qPCR, were shown in Fig. 5. It can be seen that in each reactor, the copy number of amoA was negligible in comparison with Amox and NirS, suggesting that the effect of nitrification is insignificant. In reactors SP-1, CW-1 and CW-2, both copy number and relative abundance of Amox were much higher than NirS, which indicated that anammox are more dominant than denitrification in these three reactors. On the contrary, the copy numbers of Amox and NirS were almost the same in reactor SP-2. In comparison with SP-1, it could be deduced that with higher TOC/TN level, the effects of denitrification increased for sewage plant sludge incubated reactors. For reactor CW-1 and CW-2, the same phenomenon was observed. Moreover, the copy number and relative abundance of anammox 16S RNA (Amox) gene in the CW inoculum were much higher than in the SP inoculum. Besides, as shown in point 3.1, when the TOC/TN ratio up
3.4. Microbial analysis at the end of the cultivation phase As no anammox reaction was observed over the entire operational period in reactors SP-3 and CW-3, those two reactors were thus excluded from the subsequent sequencing analysis. A phylogenetic tree showing the affiliation of anammox bacterial 16S rRNA gene sequences
Fig. 3. The consumption ratios of RS (■) and RP (
) in reactor SP-1 (a), SP-2 (b), CW-1 (c) and CW-2 (d). 458
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Fig. 4. Phylogenetic tree showing the affiliation of anammox bacterial 16S rRNA gene sequences in the reactors.
regulated from 0.0375 to 0.1, the removal efficiency of ammonium in the CW group increased by 19%, while the one in SP group increased by only 7%. All these results suggest that CWs could be a better source for the enrichment of organotrophic anammox bacteria. It is probably due to the nature of CWs. The CW in a nature-based solution can attenuate and assimilate pollutants from a wide range of wastewater streams. Moreover, in CWs, organic matter is continuously released and the TOC/TN ratio is relatively low. In the previous about Bai shiyi CW sediment, we found the existence of organotrophic anammox bacteria (Candidatus Brocadia fulgida and Candidatus anammoxoglobus propionicus), the abundances of these two kinds of anammox bacteria are 0.25% and 0.11% respectively (Zhai et al., 2016). The contribution of anammox to total nitrogen loss was about 20% (unpublished data). Besides, Gao et al. (2018) reported that in wetland ecosystems, contribution of anammox to total nitrogen can reach 41% at most. Feng et al. (2018) found that with the TOC/TN ratio changed from 0.0 to 0.1, both the copy numbers of anmmox functional gene and NirS increased. Shu et al. (2016) also reported that when the TOC/TN ratio upregulated from 0.0 to 0.1, there was a big increase in the copy number of NirS (from about 1.0 e7 to 8.0 e7). Our findings are consistent with these studies.
anammox reaction (0.11). The level of RP value indicated the occurrence of organotrophic anammox reaction. With the adjusted cycle time (6 h), the start-up time of anammox was shorten to 40 days, which is a significant progress in comparison with the results (81–145 d) reported in other studies (Lu et al., 2018; Qin et al., 2017; Wang et al., 2017a). Furthermore, the sequencing results showed that the anammox bacteria in the studied SBR had 99% similarity with Candidatus Brocadia caroliniensis, one of those reported to be capable of oxidizing VFAs. The successful enrichment of organotrophic anammox bacteria in the autocontrolled SBR proved that the ratio TOC/TN = 0.1, CW sediment as the inoculum source and no addition of chloramphenicol were the appropriate conditions for the enrichment of organotrophic anammox bacteria. 4. Conclusions The results of the batch tests showed that the TOC/TN ratio of 0.1 is appropriate value for the enrichment of organotrophic anammox bacteria (Candidatus Brocadia). The organotrophic anammox bacteria seeded from the CW sediment had a higher tolerance of acetate in comparison with the municipal sludge, suggesting that the CW sediment would be a viable alternative for the enrichment of organotrophic anammox bacteria. The start-up of organotrophic anammox process was successfully achieved in 40 days using CW sediment as inoculum and keeping the TOC/TN ratio at o.1 in an SBR reactor. The continuous addition of 50 mg/L chloramphenicol completely stopped the growth of anammox bacteria in our study, although further research is needed to conclude the inhibition effects of chloramphenicol on anammox activities.
3.5. Confirmation of the optimal enrichment of organotrophic anammox bacteria At the final stage of the study, the 5L SBR was seeded with the CW sediment and the TOC/TN ratio was set at 0.1. After 3 months of incubation, the color of the biofilm turned to red. The monitoring results of N compounds, RS and RP ratios are shown in Fig. 6. It can be seen, that ammonium and nitrite consumption began from day 10. From day 40 to day 112, the effluent nitrite was close to 0 mg/L and the effluent ammonium was about 60–85 mg/L. The RS value was 1.53 ± 0.2 (close to the theoretical value), which indicated that the anammox reaction was successfully initiated. The RP value was 0.068 ± 0.018, which was much lower than the theoretical value for autotrophic
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. Fig. 5. The copy numbers (a) and relative abundances (b) of Amox (■), NirS ( ) and amoA ( ) genes in reactor SP-1, SP-2, CW-1 and CW-2 at the end of phase I quantified by qPCR.
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Fig. 6. Performance of the SBR in terms of: a) effluent (Eff) and influent (inf) ammonium, nitrite and nitrate concentration; b) The consumption ratios of RS (■) and RP ( ) in the SBR.
Acknowledgements
anaerobic sludge blanket reactors. Bioresour. Technol. 193, 408–414. Lu, Y.F., Ma, L.J., Ma, L., Shan, B., Chang, J.J., 2018. Improvement of start-up and nitrogen removal of the anammox process in reactors inoculated with conventional activated sludge using biofilm carrier materials. Environ. Technol. 39, 59–67. Maeda, K., Toyoda, S., Philippot, L., Hattori, S., Nakajima, K., Ito, Y., et al., 2017. Relative contribution of nirK- and nirS- bacterial denitrifiers as well as fungal denitrifiers to nitrous oxide production from dairy manure compost. Environ. Sci. Technol. 51, 14083–14091. Miao, Y., Zhang, L., Yang, Y., Peng, Y., Li, B., Wang, S., et al., 2016. Start-up of singlestage partial nitrification-anammox process treating low-strength swage and its restoration from nitrate accumulation. Bioresour. Technol. 218, 771–779. Mulder, A., Van de Graaf, A.A., Robertson, L.A., et al., 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16 (3), 177–183. Myrold, D.D., Posavatz, N.R., 2007. Potential importance of bacteria and fungi in nitrate assimilation in soil. Soil Biol. Biochem. 39, 1737–1743. Park, H., Brotto, A.C., van Loosdrecht, M.C.M., Chandran, K., 2017. Discovery and metagenomic analysis of an anammox bacterial enrichment related to Candidatus “Brocadia caroliniensis” in a full-scale glycerol-fed nitritation-denitritation separate centrate treatment process. Water Res. 111, 265–273. https://doi.org/10.1016/j. watres.2017.01.011. Qin, Y., Han, B., Cao, Y., Wang, T., 2017. Impact of substrate concentration on anammoxUBF reactors start-up. Bioresour. Technol. 239, 422–429. Shu, D., He, Y., Yue, H., Gao, J., Wang, Q., Yang, S., 2016. Enhanced long-term nitrogen removal by organotrophic anammox bacteria under different TOC/TN ratio constraints: quantitative molecular mechanism and microbial community dynamics. RSC Adv. 6, 87593–87606. Sun, F., Su, X., Kang, T., Wu, S., Yuan, M., Zhu, J., Wu, W., 2016. Integrating landfill bioreactors, partial nitritation and anammox process for methane recovery and nitrogen removal from leachate. Sci. Rep. 6, 27744. https://doi.org/10.1038/ srep27744. Tao, Y., Gao, D.-W., 2012. Impact of ecological factors on anaerobic ammonia-oxidizing bacteria enrichments. Environ. Eng. Sci. 29, 479–485. Toh, S.K., Webb, R.I., Ashbolt, N.J., 2002. Enrichment of autotrophic anaerobic ammonium-oxidizing consortia from various wastewaters. Microb. Ecol. 43, 154–167. Van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S., Kuenen, J.G., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142 (8), 2187–2196. Wang, G., Xu, X., Zhou, L., Wang, C., Yang, F., 2017a. A pilot-scale study on the start-up of partial nitrification-anammox process for anaerobic sludge digester liquor treatment. Bioresour. Technol. 241, 181–189. Wang, Y., Bu, C.N., Kang, Q., Ahmad, H.A., Zhang, J., Gao, B., et al., 2017b. Autoclaved sludge as the ideal seed to culture anammox bacteria: reactor performance and microbial community diversity. Bioresour. Technol. 244, 391–399. Wang, T., Zhang, H., Gao, D., Yang, F., Zhang, G., 2012. Comparison between MBR and SBR on Anammox start-up process from the conventional activated sludge. Bioresour. Technol. 122, 78–82. Winkler, M.K., Yang, J., Kleerebezem, R., Plaza, E., Trela, J., Hultman, B., et al., 2012. Nitrate reduction by organotrophic Anammox bacteria in a nitritation/anammox granular sludge and a moving bed biofilm reactor. Bioresour. Technol. 114, 217–223. Xu, J.J., Cheng, Y.F., Xu, L.Z., Liu, Y.Y., Zhu, B.Q., Fan, N.S., et al., 2019. The revolution of performance, sludge characteristics and microbial community of anammox biogranules under long-term NiO NPs exposure. Sci. Total Environ. 649, 440–447. https://doi.org/10.1016/j.scitotenv.2018.08.386. Zhai, J., Rahaman, M.H., Chen, X., Xiao, H., Liao, K., Li, X., et al., 2016. New nitrogen removal pathways in a full-scale hybrid constructed wetland proposed from highthroughput sequencing and isotopic tracing results. Ecol. Eng. 97, 434–443. Zhang, Z.Z., Cheng, Y.F., Liu, Y.Y., Zhang, Q., Zhu, B.Q., Jin, R.C., 2019b. Deciphering the evolution characteristics of extracellular microbial products from autotrophic and mixotrophic anammox consortia in response to nitrogen loading variations. Environ. Int. 124, 501–510. https://doi.org/10.1016/j.envint.2018.12.034. Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Okabe, S., 2017. Maximum specific growth rate of anammox bacteria revisited. Water Res. 116, 296–303. Zhang, Q.Q., Zhao, Y.H., Wang, C.J., Bai, Y.H., Wu, D., Wu, J., et al., 2019a. Expression of the nirS, hzsA, and hdh genes and antibiotic resistance genes in response to recovery of anammox process inhibited by oxytetracycline. Sci. Total Environ. 681, 56–65. https://doi.org/10.1016/j.scitotenv.2019.04.438.
National Natural Science Foundation of China (grant no. 51878093 and 51478062), and the Basic and Advanced Technology Research Program of Chongqing (grant no. cstc2015jcyjBX0111) supported this research. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoleng.2019.07.039. References Cao, Y., van Loosdrecht, M.C., Daigger, G.T., 2017. Mainstream partial nitritation-anammox in municipal wastewater treatment: status, bottlenecks, and further studies. Appl. Microbiol. Biotechnol. 101, 1365–1383. Castro-Barros, C.M., Jia, M., van Loosdrecht, M.C.M., Volcke, E.I.P., Winkler, M.K.H., 2017. Evaluating the potential for dissimilatory nitrate reduction by anammox bacteria for municipal wastewater treatment. Bioresour. Technol. 233, 363–372. Chen, Q.Q., Chen, H., Zhang, Z.Z., Guo, L.X., Jin, R.C., 2017. Effects of thiocyanate on granule-based anammox process and implications for regulation. J. Hazard. Mater. 321, 81–91. https://doi.org/10.1016/j.jhazmat.2016.08.044. Coban, O., Kuschk, P., Kappelmeyer, U., Spott, O., Martienssen, M., Jetten, M.S., et al., 2015. Nitrogen transforming community in a horizontal subsurface-flow constructed wetland. Water Res. 74, 203–212. 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. Enzyme Microb. Technol. 40 (4), 859–865. Ding, C., Enyi, F.O., Adrian, L., 2018. Anaerobic Ammonium Oxidation (Anammox) with planktonic cells in a redox-stable semicontinuous stirred-tank reactor. Environ. Sci. Technol. 52 (10), 5671–5681. Ding, S., Wu, J., Zhang, M., Lu, H., Mahmood, Q., Zheng, P., 2015. Acute toxicity assessment of ANAMMOX substrates and antibiotics by luminescent bacteria test. Chemosphere 140, 174–183. Fernandez, I., Mosquera-Corral, A., Campos, J.L., Mendez, R., 2009. Operation of an Anammox SBR in the presence of two broad-spectrum antibiotics. Process Biochem. 44 (4), 494–498. Gao, D., Wang, X., Liang, H., Wei, Q., Dou, Y., Li, L., 2018. Anaerobic ammonia oxidizing bacteria: ecological distribution, metabolism, and microbial interactions. Front. Environ. Sci. Eng. 12 (3). https://doi.org/10.1007/s11783-018-1035-x. Huang, X.-L., Gao, D.-W., Tao, Y., Wang, X.-L., 2014. C2/C3 fatty acid stress on anammox consortia dominated by Candidatus Jettenia asiatica. Chem. Eng. J. 253, 402–407. Jin, R.-C., Yang, G.-F., Yu, J.-J., Zheng, P., 2012. The inhibition of the Anammox process: a review. Chem. Eng. J. 197, 67–79. https://doi.org/10.1016/j.cej.2012.05.014. Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb, R.I., et al., 2007. Candidatus “Anammoxoglobus propionicus” a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30, 39–49. Kartal, B., de Almeida, N.M., Maalcke, W.J., Op den Camp, H.J., Jetten, M.S., Keltjens, J.T., 2013. How to make a living from anaerobic ammonium oxidation. FEMS Microbiol. Rev. 37, 428–461. Koney, P., Morse, A., 2009. Effect of analgesics on the antibiotic resistance of ammoniaoxidizing organisms A paper submitted to the Journal of Environmental Engineering and Science. Can. J. Civ. Eng. 36, 1558–1566. Kumar, M., Lin, J.G., 2010. Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal–Strategies and issues. J. Hazard. Mater. 178, 1–9. Law, Y., Swa Thi, S., Chen, X.M., Nguyen, T.Q.N., Seviour, T.W., Williams, R.B.H., Wuertz, S., 2017. The start-up of mainstream anammox process is limited only by nitrite supply. Front. Wastewater Treatment Model. 4, 18–21. https://doi.org/10. 1007/978-3-319-58421-8_3. Liang, Y., Li, D., Zhang, X., Zeng, H., Yang, Y., Zhang, J., 2015. Nitrate removal by organotrophic anaerobic ammonium oxidizing bacteria with C2/C3 fatty acid in upflow
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A fast start-up of the organotrophic anammox process inoculated with constructed wetland sediment
T
Xuejiao Yina, Jun Zhaia, , Wei Hua, Yue Lia, Md. Hasibur Rahamana,c, Jacek Mąkiniab ⁎
a
Chongqing University, Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Chongqing 400045, China Department of Sanitary Engineering, Gdańsk University of Technology, 80-233 Gdansk, Poland c Department of Environmental Science and Technology, Jessore University of Science and Technology, Jessore, 7408, Bangladesh b
ARTICLE INFO
ABSTRACT
Keywords: Anammox Organotrophic bacteria TOC/TN ratio Chloramphenicol Wetland sediment qPCR
Organotrophic anaerobic ammonium oxidation (anammox) bacteria can utilize small volatile fatty acids with nitrate as electron acceptors with less energy consumption and no biomass production. To achieve a faster and stable start-up of organotrophic anammox process, in this study, the growth of organotrophic anammox bacteria seeded from hybrid constructed wetland (CW) sediment under different TOC/TN ratios and different chloramphenicol concentrations were investigated. The incubation study was conducted at the TOC/TN ratio = 0.0375–0.1 or 0.1–0.2 for the period of over five months by using serum bottles. The anammox bacteria revealed a higher activity when the TOC/TN ratio was 0.1, with the removal efficiency of NH4+-N (60–80%) and NO2−-N (~100%). The relative abundances of anammox in the incubated CW sediment were about 30% higher in comparison with the municipal waste water treatment plant sludge, suggesting the CW sediment could be a viable source for the enrichment of organotrophic anammox bacteria. On the contrary, the continuous addition of 50 mg/L chloramphenicol completely inhibited the anammox activity in our study. Following the results of the batch tests, Candidatus Brocadia caroliniensis was successfully enriched with the CW sediment in an autocontrolled SBR for the period of 40 days.
1. Introduction Anaerobic ammonium oxidation (anammox), firstly reported by Mulder et al. (1995), is one of the most important processes in the nitrogen cycle in both natural and engineered systems. One of the main drawbacks for practical applications of the anammox process is the long start-up period due to a very low specific growth rate of anammox bacteria, e.g. 0.014–0.09 d−1 (Zhang et al., 2017; Wang et al., 2017b; Cao et al., 2017). Activated sludge of municipal waste water treatment plant (WWTP) has commonly been used as the inoculum source, but there are studies indicating that also aerobic granular sludge, anaerobic granular sludge, and river or lake sediment could be used for the enrichment of anammox bacteria (Law et al., 2017; Wang et al., 2012). The choice of appropriate seeding sludge may be crucial for the effective enrichment of anammox bacteria and shortening the start-up period. For many years, anammox bacteria have been thought to be obligate autotrophic microorganisms which convert ammonia to nitrogen gas with nitrite as electron acceptor, coupled with the production of nitrate (Winkler et al., 2012; Xu et al., 2019). However, results of several
⁎
recent studies suggest that certain anammox species are capable of oxidizing short-chain volatile fatty acids (VFA), such as formate, acetate, and propionate, via the dissimilatory nitrate reduction pathway (without biomass production), followed by the normal anammox process (Kartal and de Almeida, 2013; Kumar and Lin, 2010; Winkler et al., 2012). The organotrophic nature of anammox would be advantageous due to consumption of nitrate and decreased production of surplus sludge. However, the actual ability of these organotrophic anammox species to oxidize VFA still remains unclear. In general, an adequate total organic carbon to total nitrogen (TOC/TN) ratio is potentially a key factor for their enrichment. Huang et al. (2014) observed that Candidatus Jettenia asiatica were capable of growing at the TOC/TN ratio of 0.1 (adjusted by sodium acetate) and 0.22 (adjusted by sodium propionate). With the addition of acetate, the abundance of Candidatus Jettenia, Candidatus Kuenenia and Candidatus Brocadia respectively increased, decreased significantly and decreased less, suggesting that the presence of VFA may favor the growth of Candidatus Jettenia (Liang et al., 2015). In contrast, Shu et al. (2016) showed that Candidatus Brocadia sinica had higher specific growth rates than Candidatus Jettenia asiatica and
Corresponding author. E-mail address: [email protected] (J. Zhai).
https://doi.org/10.1016/j.ecoleng.2019.07.039 Received 15 May 2019; Received in revised form 29 July 2019; Accepted 31 July 2019 Available online 31 August 2019 0925-8574/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 Inoculum sources, TOC/TN ratios and chloramphenicol concentrations used for the experiments. Group
Reactor
Inoculum source
TOC/TN
Chloram-phenicol (mg/L)
Phase I
Phase II
Ammonium (mg N/L)
Nitrite (mg N/L)
Phase I
Phase II
Phase I
Phase II
1
SP-1 SP-2 SP-3
SP* SP* SP*
0.0375 0.1 0.1
0.1 0.2 0.1
0 0 50
70–240 70–190 70–190
240 190 190
70–190 70–115 70–115
190 115 115
2
CW-1 CW-2 CW-3
CW* CW** CW**
0.0375 0.1 0.1
0.1 0.2 0.1
0 0 50
70–240 70–190 70–190
240 190 190
70–190 70–115 70–115
190 115 115
* Sewage treatment plant sludge (Ji Guanshi). ** Constructed wetland sediment (Bai Shiyi).
septic tank (450 m3), two parallel sedimentation ponds (200 m3 each), a lagoon (640 m3), five parallel vertical baffle flow wetlands (708 m2 total) in parallel, five parallel horizontal baffle flow wetlands (HSFWs) (2740 m2 total) in parallel, and a clean water pond (340 m3). In this study, the inoculum biomass was withdrawn from the HSFW sediments, where the COD/TN ratio was approximately 1.0 and the depth was 10 cm.
Candidatus Kuenenia stuttgartiensis in a wide range of the TOC/TN ratio (0.05 to 0.8). Furthermore, the organotrophic anammox process was found to be the main contributor to the TN loss at the TOC/TN ratio of 0.2. The anammox enrichment process may also be faster when the competitive bacteria are inhibited without negatively affecting anammox bacteria. There are studies (Koney and Morse, 2009; Maeda et al., 2017; Myrold and Posavatz, 2007; Tao and Gao, 2012) indicating that antibiotics, such as chloramphenicol (0.07 mM), amoxicillin (0.1–1.0 mM) and oxytetracycline (< 0.5 mM), can indeed inhibit the activity of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) or denitrifying heterotrophic bacteria (DNB), thus favoring the enrichment of anammox bacteria. Furthermore, chloramphenicol was proved to be helpful for the enrichment of Candidatus Brocadia anammoxidans (Toh et al., 2002). However, the effect of chloramphenicol on anammox bacteria in the presence of organic carbon has not been investigated. CWs have the advantage of occupying less area and being more economic. CWs are proper for the treatment of wastewater in towns. If organotrophic anammox process can be used in CWs, there will be an increase in the removal efficiency of nitrate. For applications of organotrophic anammox in CWs, it is necessary to investigate the start-up of organotrophic anammox in CWs. So in this study, we investigate the start-up of organotrophic anammox seeded from CW sediment. The start-up of organotrophic anammox seeded from sewage plant was also conducted as comparison. In the previous study at the Bai Shiyi CW, Zhai et al. (2016) reported the presence of Candidatus Brocadia fulgida and Candidatus anammoxoglobus propionicus, and these anammox species may indeed be capable of oxidizing VFA (Castro-Barros et al., 2017; Kartal et al., 2007). To our knowledge, there have been no studies showing that the CW sediment could be used as the anammox inoculum source. The effects of TOC/TN ratio (0.0375–0.1 vs. 0.1–0.2) and chloramphenicol (none vs. 50 mg/L) on organotrophic anammox were also investigated. The investigation of the chloramphenicol effect on anammox enrichment in the presence of organic carbon is another novel aspect of this study.
2.2. Batch reactor setup and operational conditions Six serum bottles (Shuniu, China) with the capacity of 200 ml each were used as batch reactors to carry out measurements of the capacity of nitrogen removal and investigate the optimum growth conditions of organotrophic anammox bacteria (described in point 2.3). The batch reactors were divided into two groups of three bottles in terms of the origin of the inoculum biomass. In group 1 (denoted as SP), the reactors were incubated with the activated sludge, whereas in group 2 (denoted as CW), the reactors were incubated with the CW sediment after screening on a sieve with apertures of 300 μm. The batch reactors were operated in a 24 h-cycle in a sequencing fed-batch mode. The operation stage of each cycle included four periods: about 23 h and 15 min of an anaerobic reaction in a shaker with the speed of 180 rpm and temperature of 33 °C, followed by a settling period of 20 min. Then the supernatant with half of the reactor liquid volume was withdrawn and replaced by fresh medium (about 3 min). Finally, the reactors were blown through with carbon dioxide (for 2 min) to adjust the pH to 7.4 ± 0.2 and then purged of dissolved oxygen by blowing nitrogen gas for 20 min. 2.3. TOC/TN ratios, chloramphenicol doses setting and nutrients in the fresh medium Different TOC/TN ratios (adjusted by addition of sodium acetate) and chloramphenicol doses set for the reactors were shown in Table 1. There were two operational phases for reactor SP-1, SP-2, CW-1 and CW-2. In phase I (day 0–136), termed the cultivation phase, a stable anammox reaction was achieved. In phase II (day 137–201), termed the experimental phase, factors influencing the growth of anammox bacteria were investigated. In the two phases, the ammonium and nitrite concentrations in the fresh medium were also shown in Table 1. In addition to ammonium and nitrite, the fresh medium also contained 500 mg/L NaHCO3, 300 mg/L MgSO4·7H2O, 180 mg/L CaCl2·2H2O, 56.7 mg/L KH2PO4, 1 ml/L trace elements I and 1 ml/L trace elements II. The content of both trace elements was prepared as recommended by Van de Graaf et al. (1996).
2. Material and methods 2.1. Origin of the inoculum biomass For experiments, the inoculum biomasses comprised activated sludge from Ji Guanshi SP of Chongqing (China) and sediment from Bai Shiyi CW of Chongqing (China). The Ji Guanshi SP treats approximately 1.2 × 106 m3/d of municipal wastewater using the A2/O process configuration in the biological stage. The inoculum biomass was withdrawn from the anaerobic compartment, where the COD/TN ratio was approximately 8.0. The Bai Shiyi CW is a hybrid system receiving municipal wastewater. The plant went into operation in January 2011, treating 500–600 m3/d of wastewater discharged from a community of approximately 6000 inhabitants. The hybrid CW plant consists of a
2.4. Confirmation of the optimal enrichment of organotrophic anammox bacteria In order to verify the findings obtained from the serial reactors with 455
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respect to the optimum TOC/TN and preferable inoculum, an autocontrolled sequencing batch reactor (SBR) with a working volume of 5 L was operated to enrich organotrophic anammox bacteria at the final stage of the study. The influent characterization was kept the same as in the optimum batch reactor and the cycle time was shortened to 6 h.
Statistics 20 (IBM, USA). 3. Results and discussion In reactor SP-1, CW-1, SP-2 and CW-2, both ammonium and nitrite were efficiently removed under non-aerated conditions, suggesting the presence of active anammox reaction in the four batch reactors without the addition of chloramphenicol. The behavior of ammonium, nitrite and nitrate removal capacities during the two operational phases (cultivation and experimental) in these four reactors was showed in Fig. 1. The removal efficiencies of ammonium and nitrite in these reactors can be seen in Fig. S1. However, with the addition of 50 mg/L of chloramphenicol over the period of five months, the removal of ammonium and nitrite during one reaction cycle was hardly observable in reactors SP-3 and CW-3 (Fig. S2). In our study, the continuous addition of chloramphenicol in the presence of organic carbon inhibited the activities of organotrophic anammox bacteria, as well as heterotrophic denitrifiers. Similar results were obtained during the operation of an anammox SBR and an anammox semi-CSTR with planktonic cells in presence of chloramphenicol (Ding et al., 2018; Fernandez et al., 2009). A concentration of 20 mg/L of chloramphenicol was continuously added to an anammox SBR causing a decrease of the specific anammox activity of the biomass from 0.25 to 0.05 gN (gVSS d−1). Ding et al. (2018) showed that anammox bacteria (Candidatus Kuenenia stuttgartiensis) were sensitive to sulfide (IC50 = 5uM) and chloramphenicol (IC50 = 19 mg/L). Jin et al. (2012) also reported that continuously addition of 20 mg/L of chloramphenicol would cause a decrease of 25% anammox effect. Except for chloramphenicol, Zhang et al. (2019) reported that after a long term of 2 mg/L oxytetracycline exposure, the anammox activity decreased by about 60%. Chen et al. (2017) reported that 130 mg/L thiocyanate could decrease the nitrogen removal efficiency by 26% within two days in the anammox reactor. However, some previous studies reported that a concentration of 100 mg/L of chloramphenicol was functional on repressing denitrifiers without inhibiting the activities of autotrophic anammonx bacteria (Toh et al., 2002), and the estimated IC50 value of chloramphenicol on the anammox process assessed by luminescent bacteria test was 409.9 mg/L after the exposure for 15 min (Ding et al., 2015). Moreover, Dapena-Mora et al. (2007) reported that chloramphenicol with concentrations up to 1 g/L did not inhibit the activity of Candidatus Kuenenia stuttgartiensis within the exposure for 6 h. Short periods of the exposure to chloramphenicol do not cause explicit inhibition to anammox activities. However, it is not ready to derive an unambiguous conclusion on the inhibition effect of chloramphenicol to anammox bacteria, and further, more systematic researches are still needed.
2.5. Analytical methods 10 ml samples (supernatant) were collected at the beginning and the end of each reaction cycle and filtrated by 0.45 μm filter membranes (Jin Teng, China). Then the samples were analyzed for ammonium, nitrite and nitrate concentrations. The measurements were performed using colorimetric methods by UV/VIS spectrophotometer (UV-2550, Shimadzu, Japan). The TN concentrations were estimated as a sum of those three N species. The TOC concentrations were detected by TOC analyzer (Shimadzu TOC-L machine, Japan). 2.6. Microbial analysis 2.6.1. DNA extraction and sequencing 5 ml samples were withdrawn from reactor SP-1, SP-2, CW-1 and CW-2 at the end of phase I. The microbial genome DNA was extracted from the samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the manufacturer’s protocol. The sequencing procedures were conducted as described by Wang et al. (2017a) and more details can be found in the Supporting Information (SI). The sequencing results were blasted on National Center for Biotechnology Information (NCBI). Then a consensus tree was built based on the maximum likelihood. The bar represented 5% estimated sequence divergence. All sequence descriptions followed the NCBI GenBank accession number. 2.6.2. Real-time quantitative polymerase chain reaction (qPCR) In order to evaluate the abundance of nitrogen removal related bacteria at the end of phase I, genomic DNA was extracted from 5 ml samples in the reactors and nitrogen transforming genes including anammox 16S RNA (Amox) gene, ammonia oxidation function gene (amoA) and denitrification function gene (NirS) were quantified by using qPCR. The qPCR was performed in a 15 μl reaction system containing 7.5 μl SYBR Green Premix (Genstar, China), 0.3 μl of forward and reverse primer, 0.3 μl of genomic DNA and 6.6 µl ddH2O. The primer information for qPCR was listed in Table 2. The R2 value for all the standard curve lines were higher than 0.99. The Ct value (threshold cycle) was determined to quantify the copy numbers of all studied genes, and duplexed assays were run in triplicate for each reaction. 2.6.3. Statistical analysis The absolute and relative abundances of anammox 16sRNA gene and two functional genes, namely nirS and amoA, were used in stepwise regression analyses with SPSS Statistics 20 (IBM, USA). A statistical One-Way ANOVA was also applied to analyze these results by SPSS
3.1. Achieving a stable anammox process in the cultivation phase (phase I) Within phase I, there was nearly no anammox reaction during the first two months, and the consumption of ammonium and nitrite was very unstable. At the beginning, a portion of nitrite was consumed while the concentrations of ammonium increased slightly. This implicitly occurred due to the combined effects of denitrifying bacteria, anammox bacteria and decay of bacteria that could not adapt to the new environment. As the system stabilized, the anammox reaction appeared around day 60 and became more prominent afterwards. For reactor SP-1 and CW-1, from day 117 on, the removal efficiencies of ammonium and nitrite stabilized over 63% and 96%, respectively. At the end of phase I, over 80 mg N/L of ammonium and 100 mg N/L of nitrite were removed in one reaction cycle. For reactor SP-2, the removal efficiencies of ammonium and nitrite reached 60% and 98% respectively after 89 days. At the end of phase I, approximately 60 mg N/L of ammonium and 90 mg N/L of nitrite were removed in one reaction cycle. For reactor CW-2, from day 75 on, the removal efficiencies of ammonium and nitrite reached 63% and 82%,
Table 2 Sequences, annealing temperatures and target genes of the primers used in this study. Primer
Sequence (5′–3′)
Annealing temp (°C)
Target gene
References
338F 820R
ACTCCTACGGGAGGCAGCA TTCGCAATGCCCGAAAGG
52
Sun et al. (2016)
1F 2R
GGGGTTTCTACTGGTGGT CCCCTCKGSAAAGCCTTCTTC
55
cd3AF9 R3cd
GTSAACGTSAAGGARACSGG GASTTCGGRTGSGTCTTGA
55
Anammox 16S RNA gene Ammonia oxidation gene Nitrite reductase gene
Miao et al. (2016) Coban et al. (2015)
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Ecological Engineering 138 (2019) 454–460
Fig. 1. The performances of reactor SP-1, SP-2, CW-1 and CW-2 in terms of the removal capacity of ammonium (■) nitrite ( cycle: (a) reactor SP-1; (b) reactor SP-2; (c) reactor CW-1; (d) reactor CW-2.
) and nitrate ( ) during one reaction
nitrogen removal efficiency increased with the operation time, however, when the TOC/TN ratio increased to 0.2, the nitrogen removal efficiency decreased sharply below 20%. These results suggest that too high TOC/TN ratio had a negative effect on the growth of anammox bacteria regardless of the inoculum source. There are some other reports about the effects of TOC/TN ratio on organotrophic anammox bacteria. Liang et al. (2015) showed that organotrophic anammox bacteria could be enriched by adding acetate or propionate with the TOC/TN ratio of 0.1. Shu et al. (2016) found that at the TOC/TN ratio of 0.2, organotrophic anammox made the largest contribution to nitrogen removal in the investigated range (0.05–0.82). Zhang et al. (2019a,b) reported that at the COD/TN ratio of 0.15, mixotrophic anammox consortia showed no difference on the removal of TN comparing with autotrophic anammox consortia. The difference of the value may implicitly be related to the species of anammox bacteria present in the reactors.
respectively. With the operation time going on, the removal efficiencies of ammonium and nitrite kept increasing. At day 103, the removal efficiencies reached the maximum level. Until to the end of phase I, the removal efficiencies of ammonium and nitrite stabilized over 86% and 94%, respectively. The removal load of ammonium and nitrite were approximately 83 mg N/L of and 118 mg N/L. In general, successful start-up times of the anammox process for reactor SP-1, SP-2, CW-1 and CW-2 were 117, 89, 117 and 103 days, respectively. After start-up, the ammonium and nitrite removal efficiencies kept at the maximum level. The start-up times of anammox process observed in the present study were comparable with other studies on autotrophic anammox enrichment. The start-up time, with ammonium and nitrite removal efficiencies stabilized at the maximum level in an SBR reactor, was 101 days as reported by Wang et al. (2012). Lu et al. (2018) operated three reactors using different materials as the biofilm carriers, for which the start-up times to achieve maximum removal efficiencies of nitrogen were in the range 105–145 days. Besides, it should be noticed that when the TOC/TN ratio was 0.0375, the anammox start-up times were both 117 days. While the TOC/TN ratio was 0.1, the anammox start-up times were 89, 103 days respectively. From these results, it could be concluded that organotrophic anammox process achieved faster start-up when the TOC/TN ratio was 0.1 rather than 0.0375.
3.3. Variations in the stoichiometric ratios Stoichiometric ratios can be used as indicators of the anammox process. The theoretical stoichiometric ratios of RS (consumption of nitrite to ammonium) and RP (production ratio of nitrate to consumption of the sum of ammonium and nitrite) of anammox reaction reported by Mulder et al. (1995) were 1.32 and 0.11, respectively. As shown in Fig. 3, the stoichiometric ratios of RS and RP in reactor SP-1 between 53 and 201 days were stable at about 1.44 ± 0.08:1 and 0.059 ± 0.018, respectively. Similarly, the stoichiometric ratios of RS and RP in reactor CW-1 between 75 and 201 days were about 1.24 ± 0.14:1 and 0.067 ± 0.014, respectively. The RS values of these two reactors were close to the theoretical value (1.32:1), which indicated the dominance of the anammox reaction for nitrogen removal. With the presence of organic carbon, the RP values for the two reactors were approximately 50–60% of the theoretical value (0.11).
3.2. Anammox performance after modification of the TOC/TN ratios in the experimental phase (phase II) For reactors SP-1 and CW-1 in phase I (TOC/TN = 0.0375), the nitrogen removal efficiency increased significantly with the operation time. During phase II, in which the TOC/TN ratio was increased to 0.1, the ammonium removal efficiency of reactors SP-1 and CW-1 increased by 7% and 19%, respectively, in comparison with the end of phase I (Fig. 2). When the TOC/TN ratio was 0.1 in reactor SP-2 and CW-2, the 457
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Fig. 2. The ammonium (■) and nitrite ( ) removal efficiency in the reactors at the end of phase I and phase II. a) SP group; b) CW group. Note: 0.1* the end of experimental phase (phase II) of reactor SP-1 and CW-1; 0.1** the end of cultivation phase (phase I) of reactor SP-2 and CW-2.
The portion of nitrate could implicitly be consumed by the dissimilatory nitrate reduction pathway of anammox bacteria. The stoichiometric ratios of RS and RP for reactor SP-2 were about 1.55 ± 0.11 and 0.12 ± 0.02 between 75 and 131 days (phase I), and changed to 2.28 ± 0.5 and 0.09 ± 0.04 between 145 and 201 days (phase II). The stoichiometric ratios of RS and RP for reactor CW-2 were about 1.38 ± 0.1 and 0.11 ± 0.017 between 61 and 131 days (phase I). However, the RS value for reactor CW-2 increased from 1.42 to 3.19, whereas the RP value dropped gradually from 0.029 to nearly 0.0 in phase II. For reactor SP-2 and CW-2, the RS and RP values in phase I were close to the theoretical values, indicating the occurrence of the anammox reaction. However, in phase II, the RS value increased significantly and the RP value decreased, which indicated that the anammox reaction was overpassed by denitrification for nitrogen removal.
in the reactors was constructed based on the sequencing results, as shown in Fig. 4. The sequencing analysis revealed that the anammox bacteria in reactor SP-1, CW-1, SP-2 and CW-2 had 99% similarity with Candidatus Brocadia caroliniensis. Candidatus Brocadia caroliniensis was reported to be capable of oxidizing short-chain VFAs (Park et al., 2017). The copy numbers and relative abundances of Amox, NirS and amoA genes, quantified by qPCR, were shown in Fig. 5. It can be seen that in each reactor, the copy number of amoA was negligible in comparison with Amox and NirS, suggesting that the effect of nitrification is insignificant. In reactors SP-1, CW-1 and CW-2, both copy number and relative abundance of Amox were much higher than NirS, which indicated that anammox are more dominant than denitrification in these three reactors. On the contrary, the copy numbers of Amox and NirS were almost the same in reactor SP-2. In comparison with SP-1, it could be deduced that with higher TOC/TN level, the effects of denitrification increased for sewage plant sludge incubated reactors. For reactor CW-1 and CW-2, the same phenomenon was observed. Moreover, the copy number and relative abundance of anammox 16S RNA (Amox) gene in the CW inoculum were much higher than in the SP inoculum. Besides, as shown in point 3.1, when the TOC/TN ratio up
3.4. Microbial analysis at the end of the cultivation phase As no anammox reaction was observed over the entire operational period in reactors SP-3 and CW-3, those two reactors were thus excluded from the subsequent sequencing analysis. A phylogenetic tree showing the affiliation of anammox bacterial 16S rRNA gene sequences
Fig. 3. The consumption ratios of RS (■) and RP (
) in reactor SP-1 (a), SP-2 (b), CW-1 (c) and CW-2 (d). 458
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Fig. 4. Phylogenetic tree showing the affiliation of anammox bacterial 16S rRNA gene sequences in the reactors.
regulated from 0.0375 to 0.1, the removal efficiency of ammonium in the CW group increased by 19%, while the one in SP group increased by only 7%. All these results suggest that CWs could be a better source for the enrichment of organotrophic anammox bacteria. It is probably due to the nature of CWs. The CW in a nature-based solution can attenuate and assimilate pollutants from a wide range of wastewater streams. Moreover, in CWs, organic matter is continuously released and the TOC/TN ratio is relatively low. In the previous about Bai shiyi CW sediment, we found the existence of organotrophic anammox bacteria (Candidatus Brocadia fulgida and Candidatus anammoxoglobus propionicus), the abundances of these two kinds of anammox bacteria are 0.25% and 0.11% respectively (Zhai et al., 2016). The contribution of anammox to total nitrogen loss was about 20% (unpublished data). Besides, Gao et al. (2018) reported that in wetland ecosystems, contribution of anammox to total nitrogen can reach 41% at most. Feng et al. (2018) found that with the TOC/TN ratio changed from 0.0 to 0.1, both the copy numbers of anmmox functional gene and NirS increased. Shu et al. (2016) also reported that when the TOC/TN ratio upregulated from 0.0 to 0.1, there was a big increase in the copy number of NirS (from about 1.0 e7 to 8.0 e7). Our findings are consistent with these studies.
anammox reaction (0.11). The level of RP value indicated the occurrence of organotrophic anammox reaction. With the adjusted cycle time (6 h), the start-up time of anammox was shorten to 40 days, which is a significant progress in comparison with the results (81–145 d) reported in other studies (Lu et al., 2018; Qin et al., 2017; Wang et al., 2017a). Furthermore, the sequencing results showed that the anammox bacteria in the studied SBR had 99% similarity with Candidatus Brocadia caroliniensis, one of those reported to be capable of oxidizing VFAs. The successful enrichment of organotrophic anammox bacteria in the autocontrolled SBR proved that the ratio TOC/TN = 0.1, CW sediment as the inoculum source and no addition of chloramphenicol were the appropriate conditions for the enrichment of organotrophic anammox bacteria. 4. Conclusions The results of the batch tests showed that the TOC/TN ratio of 0.1 is appropriate value for the enrichment of organotrophic anammox bacteria (Candidatus Brocadia). The organotrophic anammox bacteria seeded from the CW sediment had a higher tolerance of acetate in comparison with the municipal sludge, suggesting that the CW sediment would be a viable alternative for the enrichment of organotrophic anammox bacteria. The start-up of organotrophic anammox process was successfully achieved in 40 days using CW sediment as inoculum and keeping the TOC/TN ratio at o.1 in an SBR reactor. The continuous addition of 50 mg/L chloramphenicol completely stopped the growth of anammox bacteria in our study, although further research is needed to conclude the inhibition effects of chloramphenicol on anammox activities.
3.5. Confirmation of the optimal enrichment of organotrophic anammox bacteria At the final stage of the study, the 5L SBR was seeded with the CW sediment and the TOC/TN ratio was set at 0.1. After 3 months of incubation, the color of the biofilm turned to red. The monitoring results of N compounds, RS and RP ratios are shown in Fig. 6. It can be seen, that ammonium and nitrite consumption began from day 10. From day 40 to day 112, the effluent nitrite was close to 0 mg/L and the effluent ammonium was about 60–85 mg/L. The RS value was 1.53 ± 0.2 (close to the theoretical value), which indicated that the anammox reaction was successfully initiated. The RP value was 0.068 ± 0.018, which was much lower than the theoretical value for autotrophic
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. Fig. 5. The copy numbers (a) and relative abundances (b) of Amox (■), NirS ( ) and amoA ( ) genes in reactor SP-1, SP-2, CW-1 and CW-2 at the end of phase I quantified by qPCR.
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Fig. 6. Performance of the SBR in terms of: a) effluent (Eff) and influent (inf) ammonium, nitrite and nitrate concentration; b) The consumption ratios of RS (■) and RP ( ) in the SBR.
Acknowledgements
anaerobic sludge blanket reactors. Bioresour. Technol. 193, 408–414. Lu, Y.F., Ma, L.J., Ma, L., Shan, B., Chang, J.J., 2018. Improvement of start-up and nitrogen removal of the anammox process in reactors inoculated with conventional activated sludge using biofilm carrier materials. Environ. Technol. 39, 59–67. Maeda, K., Toyoda, S., Philippot, L., Hattori, S., Nakajima, K., Ito, Y., et al., 2017. Relative contribution of nirK- and nirS- bacterial denitrifiers as well as fungal denitrifiers to nitrous oxide production from dairy manure compost. Environ. Sci. Technol. 51, 14083–14091. Miao, Y., Zhang, L., Yang, Y., Peng, Y., Li, B., Wang, S., et al., 2016. Start-up of singlestage partial nitrification-anammox process treating low-strength swage and its restoration from nitrate accumulation. Bioresour. Technol. 218, 771–779. Mulder, A., Van de Graaf, A.A., Robertson, L.A., et al., 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16 (3), 177–183. Myrold, D.D., Posavatz, N.R., 2007. Potential importance of bacteria and fungi in nitrate assimilation in soil. Soil Biol. Biochem. 39, 1737–1743. Park, H., Brotto, A.C., van Loosdrecht, M.C.M., Chandran, K., 2017. Discovery and metagenomic analysis of an anammox bacterial enrichment related to Candidatus “Brocadia caroliniensis” in a full-scale glycerol-fed nitritation-denitritation separate centrate treatment process. Water Res. 111, 265–273. https://doi.org/10.1016/j. watres.2017.01.011. Qin, Y., Han, B., Cao, Y., Wang, T., 2017. Impact of substrate concentration on anammoxUBF reactors start-up. Bioresour. Technol. 239, 422–429. Shu, D., He, Y., Yue, H., Gao, J., Wang, Q., Yang, S., 2016. Enhanced long-term nitrogen removal by organotrophic anammox bacteria under different TOC/TN ratio constraints: quantitative molecular mechanism and microbial community dynamics. RSC Adv. 6, 87593–87606. Sun, F., Su, X., Kang, T., Wu, S., Yuan, M., Zhu, J., Wu, W., 2016. Integrating landfill bioreactors, partial nitritation and anammox process for methane recovery and nitrogen removal from leachate. Sci. Rep. 6, 27744. https://doi.org/10.1038/ srep27744. Tao, Y., Gao, D.-W., 2012. Impact of ecological factors on anaerobic ammonia-oxidizing bacteria enrichments. Environ. Eng. Sci. 29, 479–485. Toh, S.K., Webb, R.I., Ashbolt, N.J., 2002. Enrichment of autotrophic anaerobic ammonium-oxidizing consortia from various wastewaters. Microb. Ecol. 43, 154–167. Van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S., Kuenen, J.G., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142 (8), 2187–2196. Wang, G., Xu, X., Zhou, L., Wang, C., Yang, F., 2017a. A pilot-scale study on the start-up of partial nitrification-anammox process for anaerobic sludge digester liquor treatment. Bioresour. Technol. 241, 181–189. Wang, Y., Bu, C.N., Kang, Q., Ahmad, H.A., Zhang, J., Gao, B., et al., 2017b. Autoclaved sludge as the ideal seed to culture anammox bacteria: reactor performance and microbial community diversity. Bioresour. Technol. 244, 391–399. Wang, T., Zhang, H., Gao, D., Yang, F., Zhang, G., 2012. Comparison between MBR and SBR on Anammox start-up process from the conventional activated sludge. Bioresour. Technol. 122, 78–82. Winkler, M.K., Yang, J., Kleerebezem, R., Plaza, E., Trela, J., Hultman, B., et al., 2012. Nitrate reduction by organotrophic Anammox bacteria in a nitritation/anammox granular sludge and a moving bed biofilm reactor. Bioresour. Technol. 114, 217–223. Xu, J.J., Cheng, Y.F., Xu, L.Z., Liu, Y.Y., Zhu, B.Q., Fan, N.S., et al., 2019. The revolution of performance, sludge characteristics and microbial community of anammox biogranules under long-term NiO NPs exposure. Sci. Total Environ. 649, 440–447. https://doi.org/10.1016/j.scitotenv.2018.08.386. Zhai, J., Rahaman, M.H., Chen, X., Xiao, H., Liao, K., Li, X., et al., 2016. New nitrogen removal pathways in a full-scale hybrid constructed wetland proposed from highthroughput sequencing and isotopic tracing results. Ecol. Eng. 97, 434–443. Zhang, Z.Z., Cheng, Y.F., Liu, Y.Y., Zhang, Q., Zhu, B.Q., Jin, R.C., 2019b. Deciphering the evolution characteristics of extracellular microbial products from autotrophic and mixotrophic anammox consortia in response to nitrogen loading variations. Environ. Int. 124, 501–510. https://doi.org/10.1016/j.envint.2018.12.034. Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Okabe, S., 2017. Maximum specific growth rate of anammox bacteria revisited. Water Res. 116, 296–303. Zhang, Q.Q., Zhao, Y.H., Wang, C.J., Bai, Y.H., Wu, D., Wu, J., et al., 2019a. Expression of the nirS, hzsA, and hdh genes and antibiotic resistance genes in response to recovery of anammox process inhibited by oxytetracycline. Sci. Total Environ. 681, 56–65. https://doi.org/10.1016/j.scitotenv.2019.04.438.
National Natural Science Foundation of China (grant no. 51878093 and 51478062), and the Basic and Advanced Technology Research Program of Chongqing (grant no. cstc2015jcyjBX0111) supported this research. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoleng.2019.07.039. References Cao, Y., van Loosdrecht, M.C., Daigger, G.T., 2017. Mainstream partial nitritation-anammox in municipal wastewater treatment: status, bottlenecks, and further studies. Appl. Microbiol. Biotechnol. 101, 1365–1383. Castro-Barros, C.M., Jia, M., van Loosdrecht, M.C.M., Volcke, E.I.P., Winkler, M.K.H., 2017. Evaluating the potential for dissimilatory nitrate reduction by anammox bacteria for municipal wastewater treatment. Bioresour. Technol. 233, 363–372. Chen, Q.Q., Chen, H., Zhang, Z.Z., Guo, L.X., Jin, R.C., 2017. Effects of thiocyanate on granule-based anammox process and implications for regulation. J. Hazard. Mater. 321, 81–91. https://doi.org/10.1016/j.jhazmat.2016.08.044. Coban, O., Kuschk, P., Kappelmeyer, U., Spott, O., Martienssen, M., Jetten, M.S., et al., 2015. Nitrogen transforming community in a horizontal subsurface-flow constructed wetland. Water Res. 74, 203–212. 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. Enzyme Microb. Technol. 40 (4), 859–865. Ding, C., Enyi, F.O., Adrian, L., 2018. Anaerobic Ammonium Oxidation (Anammox) with planktonic cells in a redox-stable semicontinuous stirred-tank reactor. Environ. Sci. Technol. 52 (10), 5671–5681. Ding, S., Wu, J., Zhang, M., Lu, H., Mahmood, Q., Zheng, P., 2015. Acute toxicity assessment of ANAMMOX substrates and antibiotics by luminescent bacteria test. Chemosphere 140, 174–183. Fernandez, I., Mosquera-Corral, A., Campos, J.L., Mendez, R., 2009. Operation of an Anammox SBR in the presence of two broad-spectrum antibiotics. Process Biochem. 44 (4), 494–498. Gao, D., Wang, X., Liang, H., Wei, Q., Dou, Y., Li, L., 2018. Anaerobic ammonia oxidizing bacteria: ecological distribution, metabolism, and microbial interactions. Front. Environ. Sci. Eng. 12 (3). https://doi.org/10.1007/s11783-018-1035-x. Huang, X.-L., Gao, D.-W., Tao, Y., Wang, X.-L., 2014. C2/C3 fatty acid stress on anammox consortia dominated by Candidatus Jettenia asiatica. Chem. Eng. J. 253, 402–407. Jin, R.-C., Yang, G.-F., Yu, J.-J., Zheng, P., 2012. The inhibition of the Anammox process: a review. Chem. Eng. J. 197, 67–79. https://doi.org/10.1016/j.cej.2012.05.014. Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb, R.I., et al., 2007. Candidatus “Anammoxoglobus propionicus” a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30, 39–49. Kartal, B., de Almeida, N.M., Maalcke, W.J., Op den Camp, H.J., Jetten, M.S., Keltjens, J.T., 2013. How to make a living from anaerobic ammonium oxidation. FEMS Microbiol. Rev. 37, 428–461. Koney, P., Morse, A., 2009. Effect of analgesics on the antibiotic resistance of ammoniaoxidizing organisms A paper submitted to the Journal of Environmental Engineering and Science. Can. J. Civ. Eng. 36, 1558–1566. Kumar, M., Lin, J.G., 2010. Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal–Strategies and issues. J. Hazard. Mater. 178, 1–9. Law, Y., Swa Thi, S., Chen, X.M., Nguyen, T.Q.N., Seviour, T.W., Williams, R.B.H., Wuertz, S., 2017. The start-up of mainstream anammox process is limited only by nitrite supply. Front. Wastewater Treatment Model. 4, 18–21. https://doi.org/10. 1007/978-3-319-58421-8_3. Liang, Y., Li, D., Zhang, X., Zeng, H., Yang, Y., Zhang, J., 2015. Nitrate removal by organotrophic anaerobic ammonium oxidizing bacteria with C2/C3 fatty acid in upflow
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