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Pilot study of nitrogen removal from landfill leachate by stable nitritation-denitrification based on zeolite biological aerated filter
Waste Management 100 (2019) 161–170
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Pilot study of nitrogen removal from landfill leachate by stable nitritation-denitrification based on zeolite biological aerated filter Zhenguo Chen a,b, Xiaojun Wang a,b,⇑, Xiaokun Chen a,b, Yongyuan Yang a,b, Xiaoyang Gu c a
School of Environment and Energy, South China University of Technology, Guangzhou 510006, China The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, China c Hualu Environmental Technology Co., Ltd., Guangzhou, China b
a r t i c l e
i n f o
Article history: Received 21 May 2018 Revised 23 June 2019 Accepted 14 September 2019 Available online 17 September 2019 Keywords: Landfill leachate Nitrogen removal Nitritation Zeolite Biological aerated filter Free ammonia
a b s t r a c t A pilot (about 1 m3/d) process consisting of pre-denitrification and zeolite biological aerated filter (ZBAF) was established and run for nitrogen removal of landfill leachate. The results showed that stable nitritation and denitrification was achieved for landfill leachate with removal efficiency of Chemical Oxygen Demand (CODCr), ammonium and total nitrogen (TN) of 53.2 ± 3.0%, 93.5 ± 2.4% and 74.7 ± 9.4%, respectively. Based on the ammonium adsorption equilibrium by zeolite, stable free ammonia could be maintained for inhibition of nitrite oxidizing bacteria (NOB) and dominance of ammonia oxidizing bacteria (AOB) in ZBAF, resulting in efficient nitritation with a nitrite accumulation ratio higher than 90.0% and 3 day1. High-throughput sequencing analysis an average nitrite production rate of 1.387 kg NO 2 -N m further revealed enrichment of AOB and elimination of NOB in ZBAF. Compared to two-stage anoxicoxic process, the pilot-scale process could save approximate 5000 mg/L glucose (about 3.10 US dollar/ m3) with almost similar TN removal performance. All results obtained demonstrated the feasibility of the pilot process, which might be highly promising for the nitritation and denitrification of low C/N landfill leachate in the future. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Landfill leachate usually contains high concentration of ammonium (NH+4-N) and high levels of organic matter (Sri Shalini and Joseph, 2012; Renou et al., 2008). In order to remove these pollutants, biological methods are preferred in consideration of operational ease and cost. In most cases, complete nitrification and denitrification is one of standard choices (Sun et al., 2014), which typically presents as pre-denitrification and post-nitrification process. Although most of ammonium and biodegradable organic matter can be removed by this process, energy consumption is generally high for complete nitrification of high ammonium, and extra carbon source is always needed for low carbon to nitrogen (C/N) ratio landfill leachate (Gabarró et al., 2012). Thus, more economical biological process is important. Compared to complete nitrification and denitrification, nitritation and denitrification (also called as short-cut nitrification and ⇑ Corresponding author at: Room 301, School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Center, 510006, China. E-mail address: [email protected] (X. Wang). https://doi.org/10.1016/j.wasman.2019.09.020 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
denitrification) can save approximate 25% aeration demand and about 40% carbon source (Pollice et al., 2002; Ge et al., 2015), while ANAMMOX can almost reduce 60% of aeration energy consumption and avoid addition of carbon source (Mariusz et al., 2017). However, biodegradable organic matter in landfill leachate is not conducive to ANAMMOX for nitrogen removal pretreatment. For nitritation and denitrification, organic matter can be utilized as carbon source for denitrification. Therefore, nitritation and denitrification may be more appropriate for landfill leachate pretreatment. Stable nitrite accumulation is critical for the nitritation and denitrification of landfill leachate. In order to realize nitritation, enrichment of ammonia oxidizing bacteria (AOB) and inhibition on nitrite oxidizing bacteria (NOB) should be realized in reactor. Thus, controlling strategies such as high temperature, low dissolved oxygen (DO) and free ammonia (FA) inhibition have been commonly reported (Peng and Zhu, 2006). Though the growth rate of AOB is higher than that of NOB at temperature higher than 25 °C (Hellinga et al., 1998), it is not suitable for practical operation due to extra energy needed. DO lower than 1.0 mg/L was frequently reported for achieving nitritation of landfill leachate (Canziani et al., 2006; Kulikowska and Bernat, 2013; Chen et al., 2016), while
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strict operation should be considered for low DO level and nitritation can be easily destroyed when DO is out of control. Since oxygen plays the role of electron acceptor in ammonia oxidation, low DO levels may also lead to a low ammonium removal rate as well as low biomass yield (Wang and Yang, 2004). It was reported that inhibition on AOB and NOB appeared with FA level of 10–150 mg/L and 0.1–1.0 mg/L, respectively (Anthonisen et al., 1976). As ammonium in landfill leachate is typically high, FA inhibition is another common approach for nitritation of landfill leachate (Nhat et al., 2017; Sun et al., 2014; Zhang et al., 2015). However, in many cases, FA concentration is hard to keep stable because of its gradual decrease by ammonia removal during aeration process, leading to final failure of inhibition on NOB (Wei et al., 2014) and damage of nitrite accumulation. That is to say, stable nitrite accumulation is still an obstacle by strategies above. Compared to high temperature or low DO, FA inhibition may be more available since there is no extra energy needed or substrate limit for AOB (oxygen or ammonia). However, how to maintain stable FA levels in the reactor is still a challenge. Zeolite is a superior adsorbent for ammonium, which was also reported to be biofilm carrier for enrichment of AOB (Li et al., 2013). During the ammonium adsorption, adsorption equilibrium will eventually appear and portion of ammonium remains in liquid phase (Chen et al., 2018a). Based on this, the application of zeolite for biofilm carrier has the potential for keeping appropriate FA levels and stable nitritation can be obtained by persistent FA inhibition on NOB in during high-strength ammonium wastewater treatment. This was verified by Yang et al. (2017) who found that zeolite biological aeration filter (ZBAF) could successfully realize efficient nitritaion with stable nitrite accumulation for high concentration ammonium wastewater. This finding showed ZBAF might be feasible for nitritation and denitrification of landfill leachate. However, relevant report about ZBAF application for landfill leachate treatment is scarcely reported and whether this reactor is suitable for nitritation and denitrification of landfill leachate is still unclear. In a municipal solid waste landfill plant in Suixi, Guangdong province, China, landfill leachate was disposed by two-stage anoxic-oxic process for nitrogen removal pretreatment (Fig. S1), in which large amount of glucose was added for denitrification (Table S1). Due to the high cost for carbon source, the plant looked forward to cost-effective nitrogen pretreatment process, such as nitritation and denitrification. Thus, a pilot-scale predenitrification and ZBAF process (about 1 m3/d) was established on site and carried out for landfill leachate nitrogen and organic matter removal. During this pilot study, removal of organic matter and nitrogen were investigated. In addition, nitritation performance of ZBAF was estimated and potential nitritation mechanism of landfill leachate by ZBAF was also discussed. Moreover, variations of microbial community structure of biomass on the zeolite were also analyzed to verify nitritation performance. This study intended to estimate the feasibility of this process for nitritation and denitrification of low C/N landfill leachate.
2. Materials and methods 2.1. The pilot apparatus for landfill leachate treatment The scheme of the pilot-scale apparatus used in this study were shown in Fig. 1, which contained influent tank (PE, V = 2 m3), anoxic tank (PE, V = 5 m3), settling tank (PE, V = 2 m3), buffer tank (PE, V = 1 m3), ZBAF (anticorrosive carbon steel, u0.95 4.5 m) and effluent tank (PE, V = 2 m3), respectively. Both the influent tank and anoxic tank were equipped with mechanical mixer for stirring. The ZBAF was filled with 1–2 mm natural zeolite as biofilm carrier and
the height of zeolite carrier was about 2.5 m. Air was supplied to ZBAF for aeration. Landfill leachate was firstly mixed with 1000 mg/L glucose batch by batch in the influent tank. Then it was transported to the anoxic tank mixed with ZBAF effluent reflux by stirring for denitrification. The effluent of ZBAF was controlled by adjusting valves for desired reflux ratio. After denitrification, the influent was settled in the settling tank and flowed to the buffer tank by gravity. Then it was pumped into ZBAF for nitritation and effluent flowed to the effluent tank. During the pilot study, the influent flow rate of landfill leachate and ZBAF were all controlled by adjusting the rotor flow meter. DO in ZBAF was controlled in the range of 6.0–7.4 mg/L and temperature was kept at 28.0–33.0 °C. 2.2. Landfill leachate and seeding sludge The landfill leachate used for this study was obtained from a leachate reservoir in municipal solid waste landfill plant in Suixi, Guangdong province, China. Its characteristics were listed in Table 1. According to Table 1, it was certain that total nitrogen (TN) mainly presented as ammonium in this landfill leachate. In this treatment plant, landfill leachate was treated by a twostage anoxic and oxic process for nitrogen removal with hydraulic retention time (HRT) of about 30 days. Seeding sludge was picked from one oxic tank (500 m3/d) of the local landfill leachate treatment plant for start-up of anoxic tank and ZBAF. The mixed liquor suspended solid (MLSS) of seeding sludge was about 6000– 7000 mg/L. 2.3. Experimental operation and procedure ZBAF was first run with batch influent and continuous aeration to cultivate biofilm for 15 days. After the start-up of ZBAF, the pilot study was carried out according to Table 2. The landfill leachate inflow rate increased stepwise to 30, 35, 38 and 40 L/h, respectively, and reflux ratio was reduced to increase HRT (or reduce upflow rate) of ZBAF for releasing biological inhibition by high concentration ammonium. Based on the operational condition for twostage anoxic-oxic process in the landfill leachate treatment plant (Fig. S1), in order to obtain higher nitrogen removal efficiency, 1000 mg/L glucose was added into the influent tank to increase C/N ratio for denitrification. During this study, the removal of organic matter and nitrogen pollutants by nitritation and denitrification and ZBAF nitritation performance and potential mechanism were investigated. 2.4. Chemical analysis and calculations Chemical oxygen demand (CODCr), NH+4-N, nitrite (NO 2 -N) and nitrate (NO 3 -N) were respectively detected every day by potassium dichromate reflux method, Nessler’s reagent spectrophotometer method, N-(1-Naphthalene)-Ethylenediamine dihydrocholride spectrophotometer method, ultraviolet spectrophotometer according to Chinese standard examination of water and wastewater, respectively(China, 2002). All CODCr data were corrected according to the fact that nitrite nearly exerts a CODCr of 1.1 mg O2/mg NO 2 -N (Li et al., 2014). Since organic nitrogen in landfill leachate was very low, sum of NH+4-N, NO 2 -N and NO 3 -N was chosen to represent TN in samples. The temperature and DO were both measured by a digital DO meter (HQ30d, HACH, USA) and pH of every sample was measured by a pH meter (PHS3C, INESA Scientific Instrument Co. Ltd, China). The ZBAF packing ammonium loading rate (ALR), nitrite production rate (NPR), nitrite accumulation ratio (NAR) were calculated according to Eqs. (1), (2) and (3), respectively. FA was estimated by Eq. (4) reported by Anthonisen et al. (1976).
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Fig. 1. The schematic diagram of pilot study for landfill leachate treatment: (a) Schematic diagram, (b) Site picture.
Table 1 Characteristics of landfill leachate.
*
Content
pH
CODCr (mg/L)
NH+4-N (mg/L)
NO— 2 N (mg/L)
NO— 3 N (mg/L)
TN (mg/L)
Alkalinity (mgCaCO3/L)
Conductivity (mS/cm)
Range
8.7–9.0
6140–6725
2250–3341
ND*
5.8–11.9
2345–3362
10,500–12,400
28.3–31.4
ND means no detected.
Table 2 Operational scheme of the pilot study. Day
1–4 5–9 10–13 14–16 17–24 25 26–28 29–32
Influent flow rate (L/h)
30 35 35 35 38 40 40 40
Reflux ratio
10 8.43 9 7 6.37 6 5 4
ZBAF Inflow rate (L/h)
HRT (h)
Upflow rate (m/h)
DO (mg/L)
T (oC)
330 330 350 280 280 280 240 200
5.36 5.36 5.06 6.32 6.32 6.32 7.38 8.85
0.466 0.466 0.494 0.396 0.396 0.396 0.339 0.282
6.0–7.4
28.0–33.0
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NHþ4 N effluent NHþ4 N influent ALR kg NHþ4 N=ðm3 dayÞ ¼ 24 HRT 1000 ð1Þ
NO2 Neffluent NO2 Ninfluent NPR kg NO2 N=ðm3 dayÞ ¼ 24 HRT 1000 ð2Þ
NARð%Þ ¼
NO2
FAðmg=LÞ ¼
NO2 Neffluent 100 Neffluent þ NO3 effluent
17 NHþ4 N 10pH h i 100% 14 exp 6334 þ 10pH
ð3Þ
ð4Þ
273þT
Mass balance analysis was carried out based on the input and output pollutant of each operational unit. The concentration varia tion of each pollutant DX (CODCr, NH+4-N, NO 2 -N or NO3 -N) in each operational unit was calculated according to Eq. (5) as followed.
DX ¼
X output X input Q out
ð5Þ
where Xinput and Xoutput was the total mass amount pollutant input or output per unit time of each operational unit, mg/h; Qout was the output flow rate of each operational unit, L/h. The plus or minus sign of DX represented that pollutant concentration increased or decreased in that operational unit. 2.5. Metagenomic DNA extraction and high-throughput sequencing analysis 2.5.1. Extraction of DNA from microbiological samples Seeding sludge from the oxic tank of local landfill leachate treatment plant was picked at the beginning of pilot study (named O1) and at day 30 (named O2), and bio-zeolite sample was also collected at day 10, 20, 25 and 30, named as Z1, Z2, Z3 and Z4, respectively. All microbiological samples were washed as per protocol described earlier (Chen et al., 2016) and extracted for total genomic DNA using a Bacterial DNA Isolation Kit (50, D3350-01, OMEGA, USA) according to the manufacturer’s instructions. The DNA samples concentration and quantity were all detected by ultraviolet and visible spectrophotometer at 260 and 280 nm, and Agarose Gel Electrophoreses, respectively. 2.5.2. Amplicon generation and purification The amplicon generation of 16S rRNA was performed using the specific primer 319F-806R for 16S V4 with the barcode (Yang et al., 2017) on Illumina Miseq PE250 platform. The PCR reaction was carried out with a total volume of 50 lL and 30 ng genome DNA, 4 lL of PCR Prime Cocktail, 25 lL of Phusion High-Fidelity PCR Master Mix (New England Biolans) was used. The detailed operation of PCR included initial denaturation at 98 °C for 3 min, 30 cycles of denaturation at 72 °C for 45 s; extension at 72 °C for 7 min; and finally holding at 4 °C. The PCR productions were purified with AmpureXPbeads (AGENCORT) for removing the unspecific products. 2.5.3. Statistical analysis In order to obtain more accurate and reliable results in subsequent bioinformatics analysis, the raw data was pre-processed to get clean data by in-house procedure as following: (1) Truncation of sequence reads nor having an average quality of 20 over a 30 bp sliding window based on the phred algorithm, and trimmed reads having less than 75% of their original length, as well as its paired read, will be removed; (2) Removal of reads contaminated by adapter (default parameter: 15 bases overlapped by reads and
adapter with maximal 3 bases mismatch allowed); (3) Removal of reads with ambiguous base (N base), and its paired reads; (4) Removal of reads with low complexity (default: reads with 10 consecutive same base). For pooling library with barcode samples mixed, the clean reads were assigned to corresponding samples by allowing 0 base mismatch to barcode sequences with inhouse scripts. The reads with sequencing adapter, N base, poly base, low quality etc., were filtered out with default parameters aforementioned (Table S2). If the two paired-end reads overlapped, the concensus sequence was generated by FLASH (Fast Length Adjustment of Short reads, v 1.2.11), and the detailed method is as follows: (1) Minimal overlapped length: 15 bp; (2) Mismatching ratio of overlapped region: 0.1; (3) Removal of paired-end reads without overlaps. The high quality paired-end reads were combined to tags based on overlaps. 174,873 tags were obtained in total with 29,145 tags per sample on average (Table S3), and the average length was 253 bp. The tags obtained were clustered to OTU (Operational Taxonomic Unit) by script of software USEARCH (v7.0.1090) with a 97% similarity threshold by using UPARSE, and the OTU unique representative sequences were obtained. Chimeras were filtered out by using UCHIME (v4.2.40). All tags were mapped to each OTU representative sequencing using USEARCH GLOBAL, then the tags number of each OTU in each sample was summarized to OTU abundance table. Chao, Ace, Shannon index and Simpson index was calculated for alpha diversity analysis. The Illumina high-throughput sequencing methods and data analysis were under the consideration of quality assurance and quality control. QIIME was used for analyzing the clustering of taxonomic based on OTUs. 3. Results and discussion 3.1. Performance of the pilot study Fig. 2 showed the overall performance of the pilot study for the treatment of landfill leachate. According to Fig. 2(a), influent and effluent CODCr of the pilot apparatus ranged from 6200 to 6800 mg/L and 2600 to 3300 mg/L, respectively, leading to an overall average CODCr removal efficiency of 53.2 ± 3.0%. In Fig. 2(b), from day 10 to day 24, effluent NH+4-N stayed stable at 183.7 ± 23.2 mg/L with an approximate removal efficiency of 93.5 ± 1.2%. When inflow rate increased to 40L/h, however, effluent NH+4-N increased from 200 to 300 mg/L and its removal efficiency also decreased to about 90.0%. It could be seen in Fig. 2(c) that TN removal efficiency gradually decreased along with increase of inflow rate. With inflow rate of 40 L/h, average TN removal efficiency of the pilot apparatus was about 66.0 ± 6.3% under an average ALR of 1.93 ± 0.26 kg NH+4-N m3 day1 on ZBAF. All in all, it was clear that CODCr, NH+4-N and TN were significantly removed from landfill leachate by the pilot apparatus. 3.2. Pollutants removal in different units The detailed variations of CODCr, NH+4-N, NO 2 -N, NO3 -N and pH in anoxic tank, buffer tank and ZBAF were listed in Fig. 3(a), (b), (c), (d), (e), respectively. Compared to influent for this study, CODCr ranged at 3000–3500 mg/L in anoxic tank and then slightly declined in buffer tank and ZBAF. According to Fig. 3(b) and (c), compared to anoxic tank and buffer tank, average effluent NH+4-N and NO 2 -N of ZBAF was 180.0 ± 62.1 and 499.4 ± 189.0 mg/L, respectively, showing remarkable NH+4-N decrease and NO 2 -N accumulation in ZBAF. In Fig. 3(d), average effluent NO 3 -N of anoxic tank, buffer tank and ZBAF was only 15.2 ± 5.8, 14.4 ± 6.4 and 31.9 ± 7.4 mg/L during the whole pilot study, respectively.
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(DCODCr = 430.0 mg/L) and TN (DTN = 245.2 mg/L). It should be pointed out that majority of removed nitrogen in anoxic tank was nitrite (DNO 2 -N = 208.5 mg/L), which was offered by reflux from ZBAF. These data revealed denitrification via nitrite pathway occurred in the anoxic tank indeed. Besides, a portion of CODCr and TN were also removed in buffer tank, which revealed that denitrification should also happen in buffer tank. In ZBAF, ammonium was apparently removed withDNH+4-N as 311.0 mg/L and nitrite accumulated dramatically withDNO 2 -N as 444.0 mg/L. On the contrary, Nitrate was reduced in ZBAF(DNO 3 -N = 22.8 mg/L). As ZABF was a kind of biofilm reactor, anoxic microenvironment might be easily created and then denitrification could happen with a few organic matter (Ryu et al., 2008). Since CODCr also decreased in ZBAF (DCODCr = 61.8 mg/L), it could be speculated that nitrate denitrification possibly occurred. The concentration difference between NO 2 -N and NO3 -N in the ZBAF effluent meant that NOB was deeply inhibited and excellent nitrite accumulation was achieved in ZBAF, indicating this reactor played a key role in achieving stable nitritation of the landfill leachate. In addition, TN also increased in ZBAF on day 20 (DTN = 110.2 mg/L), possibly resulting from the ammonium release of zeolite fillings. This finding meant that zeolite fillings might show an effect on nitrogen concentration balance in ZBAF by ammonium release or adsorption with influent ammonium concentration fluctuations (Yang et al., 2017). 3.3. Nitritation performance and mechanism of ZBAF
Fig. 2. The overall performance of pilot study in treating landfill leachate: (a) CODCr, (b) NH+4-N, (c) TN.
Fig. 3(e) showed pH value in effluent of anoxic tank, buffer tank and ZBAF was 9.02 ± 0.05, 9.20 ± 0.07 and 8.61 ± 0.23, respectively. In order to better understand the pollutants removal pathway in this study, analysis data on day 20 was picked as an example for mass balance analysis (Fig. 4). After mass balance calculation, pollutant concentration variations DX in each operational unit were listed in Table 3. According to mass balance analysis results, the anoxic tank was the main reactor for removal of organic matter
In order to estimate nitritation performance of ZBAF, ALR and NPR was calculated and shown in Fig. 5(a). Combined with Fig. 3, from day 1 to day 9, even though influent ALR increased simultaneously from 1.520 to 2.018 kg NH+4-N m3 day1, NPR of ZBAF kept 3 in a rising trend from 0.468 to 1.354 kg NO day1. When 2 -N m ALR increased to 2.868 ± 0.095 kg NH+4-N m3 day1 during day 10 13, significant drop of NPR happened, with lowest NPR of 3 0.691 kg NO day1. With influent upflow rate of 2 -N m 0.396 m/h during day 14–24, highest NRP appeared as about 3 1.686 kg NO day1. However, when influent inflow rate 2 -N m increased to 40 L/h after day 24, NRP decreased again even though influent ALR decreased to about 1.927 ± 0.261 kg NH+4-N m3 day1. As aforementioned, ZBAF showed efficient nitritation in this study. According to Fig. 5(b), FA in ZBAF during the whole study was typically high meanwhile NAR was basically higher than 90.0% in most of cases. Even though influent NH+4-N of ZBAF varied from 293.4 to 687.8 mg/L, effluent NH+4-N of ZBAF always kept in the range of 81.8–306.0 mg/L, which should be attributed to the frequently automatic shift of adsorption equilibrium by zeolite in ZBAF (Yang et al., 2017). Due to this phenomenon, ZBAF could maintain an appropriate FA range and also showed buffer function in spite of different influent ammonium concentration. In the literature, FA concentration specifically differed for sustainable landfill leachate nitritation and deep inhibition on NOB. Zhang et al. (2015) found that nitrite accumulation rate remained high with FA from 5.30 to 48.67 mg/L in landfill leachate treatment, while maximum FA concentration as high as 506 mg/L was also reported for nitritation of old landfill leachate (Nhat et al., 2017). In this study, influent NH+4-N for ZBAF gradually increased from day 1 to day 9 with FA range of 22.83–50.08 mg/L. During this period, NPR kept rising 3 and reached 1.354 kg NO day1 at day 9, indicating that 2 -N m this FA range showed distinct inhibition on NOB but not AOB in ZBAF. However, with upflow rate increased to 0.494 m/h during day 10–13, significant drop of NPR appeared as FA increased to 64.72–92.34 mg/L, which provided evidence that AOB was also inhibited. By decreasing upflow rate increased to 0.396 m/h, this situation changed quickly with an average NPR of 3 1.400 ± 0.231 kg NO day1 even though landfill leachate 2 -N m
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Fig. 3. Typical profiles of CODCr, nitrogen concentration and pH in different units: (a) CODCr, (b) NH+4-N, (c) NO 2 -N, (d) NO3 -N, (e) pH.
inflow rate increased to 38 L/h. These results further demonstrated that FA inhibition on AOB in ZBAF might be reversible. When inflow rate increased to 40 L/h, NPR dropped again with obvious effluent NO 2 -N decrease, which might be attributed to severe FA inhibition on AOB by range of 63.20–93.81 mg/L. Based on results above, it could be concluded that influent flow rate should decide nitritation performance of ZBAF. The total vessel volume of the whole pilot apparatus was about 9.8 m3, including anxic tank, settling tankd, buffer tank and ZBAF packings. Therefore, in order to obtain desired nitritation performance, total HRT of the process for landfill leachate should not less than 10.2 days. Since the oxygen affinity constant of AOB and NOB was 0.3– 0.5 mg/L and 0.7–1.8 mg/L, respectively (Guisasola et al., 2005), low DO concentration was chosen for nitritation. Nevertheless, DO in ZBAF was typically as high as 6.0–7.4 mg/L in this pilot study. Thus, DO could not be the main factors for inhibiting NOB and realizing stable nitritation. It was reported that growth rate of AOB was higher than that of NOB with temperature higher than 20 °C (Hellinga et al., 1998). The temperature in ZBAF varied from 28.0 to 33.0 °C, suggesting that effect of temperature on promoting nitritation should be taken into consideration. According to Fig. 3 (e), pH in ZBAF maintained at the range of 8.12–9.01 during the whole study, which could result in a higher FA value compared to the situation that pH less than 8.0 according to Eq. (4). Based on the temperature and pH value, FA of ZBAF kept in 22.84– 93.81 mg/L. This FA was higher than FA of former study ranging in 17.35–40.87 mg/L for determining excellent nitritation performance in ZBAF (Yang et al., 2017). Therefore, FA inhibition should be the main reason explaining stable and efficient nitritation in ZBAF for this pilot study. Besides, high DO levels presented no damage on nitrite accumulation in ZBAF. Instead, this DO level could offer more oxygen for metabolism of AOB to promote nitritation performance (Regmi et al., 2014). This finding showed the advantage of ZBAF for nitrtitaion of landfill leachate compared to other methods and further revealed the feasibility and superiority of ZBAF.
3.4. Microbial community taxonomic analysis The microbial community variations of biofilm on zeolite carriers compared to inoculated sludge were investigated by highthroughput sequencing technology. Table 4 listed the values of effective reads, OTUs, Chao and Shannon index of all biological samples. With the operation of the pilot-scale process, OTUs, Chao and Shannon index all increased as running time increased, showing gradual microbial diversity improvement in ZBAF. Compared O1 with O2, these results demonstrated that microbial communities in ZBAF changed. According to NMDS analysis results (Fig. 6), obvious distance between zeolite biofilm samples and sludge samples indicated the difference of community structure between ZBAF and oxic tank. In Fig. 7 taxonomic classifications of all samples were presented at phylum and class level. For the inoculated sludge (O1), three most abundant bacteria at phyla level were Bacteroidetes (46.37%), Proteobacteria (24.49%) and Thermi (8.09%), and then shifted slightly with Bacteroidetes (56.00%), Proteobacteria (23.37%) and Thermi (13.57%) after about a month operation of oxic tank. Due to the continuous operation under inhibition of FA in ZBAF, remarkable microbial community variation happened in ZBAF compared to the oxic tank, with Bacteroidetes (43.92%, 44.38%, 44.52%, 46.52%), Proteobacteria (44.38%, 43.09%, 44.06%, 41.28%) and Thermi (3.00%, 3.44%, 2.98%, 2.47%) at day 10, 20, 25, 30, respectively. The noteworthy relative abundance of Proteobacteria in ZBAF showed dominance of this phylum, which was similar to results that both of efficient NH+4-N removal and nitrite accumulation appeared with richness of Proteobacteria (Wang et al., 2014). In addition, Betaproteobacteria at class level of all ZBAF samples were higher than that of the sludge samples, which further explained the reason for nitrite accumulation in ZBAF (Gao et al., 2011; Chen et al., 2016). Besides, class Nitrospira were all detected in O1 and O2 with relative abundance of 4.07% and 1.29%, while none of this class was detected in each of zeolite biofilm samples, which revealed the washout of NOB in ZBAF.
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Fig. 4. Mass balance analysis of CODCr and nitrogen based on data of day 20.
Table 3 Pollutant concentration variations in different unit on day 20 after mass balance analysis Unit: mg/L. Unit
Anoxic tank
Buffer tank
ZBAF
DCODCr DNH+4-N DNO 2 -N DNO 3 -N DTN
430.0 19.1 208.5 17.6 245.2
107.5 5.0 135.0 2.1 142.1
61.8 311 444 22.8 110.2
In the literature, accumulation of AOB and washout of NOB were commonly reported in study of nitritation (Daims et al., 2015; Fitzgerald et al., 2015). For ZBAF, Yang et al. (2017) also found that Nitrosomonas was typically dominant in ZBAF for high
concentration ammonium wastewater treatment. In this pilot study, the relative abundance of Nitrosomonas at genus level in ZBAF was found to be 22.54%, 21.72%, 21.78% and 20.15% at day 10, 20, 25, 30, respectively, while genus Nitrospira was undetectable (Table 5). These findings definitely verified the dominance of AOB and washout of NOB by severe FA inhibition in ZBAF with FA as high as 22.83–93.81 mg/L in the pilot study. In other words, ZBAF was proved to be feasible for stably efficient nitritation of landfill leachate.
3.5. Significance of this study and issue to be addressed Based on the nitritation performance of ZBAF, this pilot study presented with pre-denitrification and post-nitritation was suc-
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Fig. 5. ALR, NPR, NAR and FA in ZBAF during the pilot study: (a) ALR and NPR, (b) NAR and FA.
Table 4 The numbers of reads and alpha diversity results.a Sample ID Reads Alpha diversity
a b c d
Raw Effective OTUsb Chaoc Shannond
O1
O2
Z1
Z2
Z3
Z4
30,076 28,956 476 502 3.732
30,650 29,573 388 439 2.864
29,990 28,937 429 461 3.519
30,041 29,082 432 466 3.585
30,307 29,344 458 520 3.602
30,413 29,482 456 535 3.701
Similarity level = 0.97. OTUs: operational taxonomic units (calculated at the 3% distance limit). Chao is a kind of alpha diversity index reflecting microbial community species richness of sample. A higher number means a higher richness. Shannon is a kind of alpha diversity reflecting microbial community species diversity of sample. A higher number represents more diversity.
Fig. 6. NMDS analysis results of different samples.
cessfully applied for organic matter and nitrogen removal from landfill leachate. During this study, average CODCr, NH+4-N and TN removal efficiency was 53.2 ± 3.0%, 93.5 ± 2.4% and 74.7 ± 9.4% 3 respectively with an average NPR of 1.387 kg NO day1. 2 -N m The operational cost for this landfill leachate was mainly decided by energy needed for aeration and dosage of extra carbon source. During the whole study, although extra carbon source (1000 mg/ L glucose, about 0.62 US dollar/m3) was added in influent, nitrite could not be removed totally in anoxic tank or buffer tank (Fig. 3 (c)) so that TN removal was not thorough. As the theoretical C/N ratio for nitrite denitrification was 1.78 (Chen et al., 2018b), higher
TN removal efficiency could be realized by increasing glucose dosage in influent. According to data in Table S1, TN removal efficiency of two-stageanoxic-oxic process in the plant was about 78.1% with 6000 mg/L glucose (about 3.72 US dollar/m3) dosed, which was only 3.4% higher than that of presented process. That was to say, leaving out energy consumed for aeration, approximate 5000 mg/L glucose (about 3.10 US dollar/m3) could be saved to achieve almost similar TN removal performance. The plant could save about 310 US dollar/day if the presented process based on ZBAF was applied for nitrogen removal pretreatment of low C/N landfill leachate. Currently, most of pretreatment process for low C/N ratio landfill leachate in China is still the traditional two-stage anoxic/oxic process, in which ammonium is converted by complete nitrification and nitrogen is removed by nitrate denitrification. In this case, many landfill leachate treatment plants are facing the situation that operational cost for landfill leachate pretreatment stays at a high level because of the extra dosage of carbon source. However, this situation can definitely change if the presented process based on ZBAF reported in this study is applied for landfill leachate pretreatment, which shows potential application prospect in saving operational cost by realizing stable nitritation and denitrification. In addition, it should be noted that both of NH+4-N and NO 2 -N existed in the effluent in a certain concentration range. From this point of view, ANAMMOX might be possible for subsequent nitrogen removal of effluent if the ratio of NH+4-N and NO 2 -N in effluent could be controlled by adjusting operational parameters of ZBAF. That is to say, a promising nitrogen removal process based on ZBAF and ANAMMOX for low C/N landfill leachate is potentially possible. Besides, the effect of potential ammonium release from zeolite in ZBAF still needed investigation in the future.
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Fig. 7. Taxonomy classification of bacterial diversity at Phylum and Class level of seed sludge and ZBAF: (a) Phylum level, (b) Class level.
Table 5 Comparison of relative abundance of AOB and NOB for different samples.
*
Sample
O1
O2
Z1
Z2
Z3
Z4
Nitrosomonas Nitrospira
3.21 3.75
2.24 1.23
22.54 ND*
21.72 ND
21.78 ND
20.15 ND
ND means no bacterium at genus level was detected.
4. Conclusions The pilot scale pre-denitrification and ZBAF process succeeded in achieving stable nitritation and denitrification for low C/N landfill leachate. During the whole operation, removal efficiency of CODCr, NH+4-N and TN of pilot scale process was 53.2 ± 3.0%, 93.5 ± 2.4% and 74.7 ± 9.4%, respectively. In this process, ZBAF was the key for stable nitrite accumulation with NAR higher than 90.0%. Based on the zeolite fillings, ZBAF showed excellent buffer function with frequent variation of influent NH+4-N concentration and maintained an appropriate FA range for inhibition on NOB, which was the main factor for efficient nitritation. Highthroughput sequencing analysis results further revealed enrichment of AOB and elimination of NOB definitely happened in ZBAF. The nitrogen removal pretreatment process based on ZBAF was highly promising for low C/N landfill leachate treatment. Acknowledgement This work was supported by the Specialized Applied Science and Technology Research, Development and Major Transformation Project of Guangdong Province in 2017 (2017B020236004). Authors are also thankful to Weilong Li and the staffs of Suixi landfill leachate treatment plant for their kindly helps. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.09.020. References Anthonisen, A.C., Loehr, R.C., Prakasam, T.B.S., Srinath, E.G., 1976. Inhibition of nitrification by ammonia and nitrous acid. Water Pollut. Control Federat. 48 (5), 835–852. Canziani, R., Emondi, V., Garavaglia, M., Malpei, F., Pasinetti, E., Buttiglieri, G., 2006. Effect of oxygen concentration on biological nitrification and microbial kinetics in a cross-flow membrane bioreactor (MBR) and moving-bed biofilm reactor (MBBR) treating old landfill leachate. J. Membr. Sci. 286 (1–2), 202–212.
Chen, Z., Wang, X., Chen, X., Chen, J., Feng, X., 2018a. Nitrogen removal via nitritation pathway for low-strength ammonium wastewater by adsorption, biological desorption and denitrification. Bioresour. Technol. 267, 541–549. Chen, Z., Wang, X., Chen, X., Chen, J., Gu, X., 2018b. Rapid start-up and performance of denitrifying granular sludge in an upflow sludge blanket (USB) reactor treating high concentration nitrite wastewater. Biodegradation 29 (6), 543–555. Chen, Z., Wang, X., Yang, Y., Mirino, M.W., Yuan, Y., 2016. Partial nitrification and denitrification of mature landfill leachate using a pilot-scale continuous activated sludge process at low dissolved oxygen. Bioresour. Technol. 218, 580–588. China, E.P.A., 2002. Standard methods of examination of water and wastewater, fourth ed. Beijing. Daims, H., Lebedeva, E.V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Jehmlich, N., Palatinszky, M., Vierheilig, J., Bulaev, A., Kirkegaard, R.H., von Bergen, M., Rattei, T., Bendinger, B., Nielsen, P.H., Wagner, M., 2015. Complete nitrification by Nitrospira bacteria. Nature 528 (7583), 504–509. Fitzgerald, C.M., Camejo, P., Oshlag, J.Z., Noguera, D.R., 2015. Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen. Water Res. 70, 38–51. Gabarró, J., Ganigué, R., Gich, F., Ruscalleda, M., Balaguer, M.D., Colprim, J., 2012. Effect of temperature on AOB activity of a partial nitritation SBR treating landfill leachate with extremely high nitrogen concentration. Bioresour. Technol. 126, 283–289. Gao, D., An, R., Tao, Y., Li, J., Li, X., Ren, N., 2011. Simultaneous methane production and wastewater reuse by a membrane-based process: evaluation with raw domestic wastewater. J. Hazard. Mater. 186 (1), 383–389. Ge, S., Wang, S., Yang, X., Qiu, S., Li, B., Peng, Y., 2015. Detection of nitrifiers and evaluation of partial nitrification for wastewater treatment: a review. Chemosphere 140, 85–98. Guisasola, A., Jubany, I., Baeza, J.A., Carrera, J.N., Lafuente, J., 2005. Respirometric estimation of the oxygen affinity constants for biological ammonium and nitrite oxidation. J. Chem. Technol. Biotechnol. 80 (4), 388–396. Hellinga, C., Schellen, A.A.J.C., Mulder, J.W., Loosdrecht, M.C.M. Van, Heijnen, J.J., 1998. The sharon process: an innovative method for nitrogen removal from ammonium-rich waste water. Water Sci. Technol. 37, 135–142. Kulikowska, D., Bernat, K., 2013. Nitritation-denitritation in landfill leachate with glycerine as a carbon source. Bioresour. Technol. 142, 297–303. Li, H., Zhou, S., Ma, W., Huang, P., Huang, G., Qin, Y., Xu, B., Ouyang, H., 2014. Longterm performance and microbial ecology of a two-stage PN-ANAMMOX process treating mature landfill leachate. Bioresour. Technol. 159, 404–411. Li, Q., Sun, S., Guo, T., Yang, C., Song, C., Geng, W., Zhang, W., Feng, J., Wang, S., 2013. Short-cut nitrification in biological aerated filters with modified zeolite and nitrifying sludge. Bioresour. Technol. 136, 148–154. Mariusz, T., Grzegorz, C., Aleksandra, Z., 2017. Influence of temperature and pH on the anammox process: a review and mata-analysis. Chemosphere 182, 203– 214. Nhat, P.T., Biec, H.N., Van, T.T.T., Van Tuan, D., Trung, N.L.H., Nghi, V.T.K., Dan, N.P., 2017. Stability of partial nitritation in a sequencing batch reactor fed with high
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ammonium strength old erban landfill leachate. Int. Biodeter. Biodegrad. 124, 56–61. Peng, Y., Zhu, G., 2006. Biological nitrogen removal with nitrification and denitrification via nitrite pathway. Appl. Microbiol. Biotechnol. 73 (1), 15–26. Pollice, A., Tandoi, V., Lestingim, C., 2002. Influence of aeration and sludge retention time on ammonium oxidation to nitrite and nitrate. Water Res. 36, 2541–2546. Regmi, P., Miller, M.W., Holgate, B., Bruce, R., Paark, H., Chandran, K., Wett, B., Murthy, S., Bott, C.B., 2014. Control of aeration, Aerobic SRT and COD input for mainstream nitritation/denitrification. Water Res. 57, 162–171. Renou, S., Givaudan, J.G., Poulain, S., Dirassouyan, F., Moulin, P., 2008. Landfill leachate treatment: review and opportunity. J. Hazard. Mater. 150 (3), 468–493. Ryu, H.D., Kim, D., Lim, H.E., Lee, S.I., 2008. Nitrogen removal from low carbon-tonitrogen wastewater in four-stage biological aerated filter system. Process Biochem. 43 (7), 729–735. Sri Shalini, S., Joseph, K., 2012. Nitrogen management in landfill leachate: application of SHARON, ANAMMOX and combined SHARON-ANAMMOX process. Waste Manage. 32 (12), 2385–2400.
Sun, F., Sun, S., Li, Q., Deng, X., Hu, J., Wu, W., 2014. Pilot-scale nitrogen removal from leachate by ex situ nitrification and in situ denitrification in a landfill bioreactor. Chemosphere 101, 77–85. Wang, J., Yang, N., 2004. Partial nitrification under limited dissolved oxygen conditions. Process Biochem. 39 (10), 1223–1229. Wang, L., Zeng, G., Yang, Z., Luo, L., Xu, H., Huang, J., 2014. Operation of partial nitrification to nitrite of landfill leachate and its performance with respect to different oxygen conditions. Biochem. Eng. J. 87, 62–68. Wei, D., Xue, X., Yan, L., Sun, M., Zhang, G., Shi, L., Du, B., 2014. Effect of influent ammonium concentration on the shift of full nitritation to partial nitrification in a sequencing batch reactor at ambient temperature. Chem. Eng. J. 235, 19–26. Yang, Y., Chen, Z., Wang, X., Zheng, L., Gu, X., 2017. Partial nitrification performance and mechanism of zeolite biological aerated filter ammonium wastewater treatment. Bioresour. Technol. 241, 473–481. Zhang, C., Zhang, S., Zhang, L., Rong, H., Zhang, K., 2015. Effects of constant pH and unsteady pH at different free ammonia concentrations on shortcut nitrification for landfill leachate treatment. Appl. Microbiol. Biotechnol. 99 (8), 3707–3713.
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Pilot study of nitrogen removal from landfill leachate by stable nitritation-denitrification based on zeolite biological aerated filter Zhenguo Chen a,b, Xiaojun Wang a,b,⇑, Xiaokun Chen a,b, Yongyuan Yang a,b, Xiaoyang Gu c a
School of Environment and Energy, South China University of Technology, Guangzhou 510006, China The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, China c Hualu Environmental Technology Co., Ltd., Guangzhou, China b
a r t i c l e
i n f o
Article history: Received 21 May 2018 Revised 23 June 2019 Accepted 14 September 2019 Available online 17 September 2019 Keywords: Landfill leachate Nitrogen removal Nitritation Zeolite Biological aerated filter Free ammonia
a b s t r a c t A pilot (about 1 m3/d) process consisting of pre-denitrification and zeolite biological aerated filter (ZBAF) was established and run for nitrogen removal of landfill leachate. The results showed that stable nitritation and denitrification was achieved for landfill leachate with removal efficiency of Chemical Oxygen Demand (CODCr), ammonium and total nitrogen (TN) of 53.2 ± 3.0%, 93.5 ± 2.4% and 74.7 ± 9.4%, respectively. Based on the ammonium adsorption equilibrium by zeolite, stable free ammonia could be maintained for inhibition of nitrite oxidizing bacteria (NOB) and dominance of ammonia oxidizing bacteria (AOB) in ZBAF, resulting in efficient nitritation with a nitrite accumulation ratio higher than 90.0% and 3 day1. High-throughput sequencing analysis an average nitrite production rate of 1.387 kg NO 2 -N m further revealed enrichment of AOB and elimination of NOB in ZBAF. Compared to two-stage anoxicoxic process, the pilot-scale process could save approximate 5000 mg/L glucose (about 3.10 US dollar/ m3) with almost similar TN removal performance. All results obtained demonstrated the feasibility of the pilot process, which might be highly promising for the nitritation and denitrification of low C/N landfill leachate in the future. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Landfill leachate usually contains high concentration of ammonium (NH+4-N) and high levels of organic matter (Sri Shalini and Joseph, 2012; Renou et al., 2008). In order to remove these pollutants, biological methods are preferred in consideration of operational ease and cost. In most cases, complete nitrification and denitrification is one of standard choices (Sun et al., 2014), which typically presents as pre-denitrification and post-nitrification process. Although most of ammonium and biodegradable organic matter can be removed by this process, energy consumption is generally high for complete nitrification of high ammonium, and extra carbon source is always needed for low carbon to nitrogen (C/N) ratio landfill leachate (Gabarró et al., 2012). Thus, more economical biological process is important. Compared to complete nitrification and denitrification, nitritation and denitrification (also called as short-cut nitrification and ⇑ Corresponding author at: Room 301, School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Center, 510006, China. E-mail address: [email protected] (X. Wang). https://doi.org/10.1016/j.wasman.2019.09.020 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
denitrification) can save approximate 25% aeration demand and about 40% carbon source (Pollice et al., 2002; Ge et al., 2015), while ANAMMOX can almost reduce 60% of aeration energy consumption and avoid addition of carbon source (Mariusz et al., 2017). However, biodegradable organic matter in landfill leachate is not conducive to ANAMMOX for nitrogen removal pretreatment. For nitritation and denitrification, organic matter can be utilized as carbon source for denitrification. Therefore, nitritation and denitrification may be more appropriate for landfill leachate pretreatment. Stable nitrite accumulation is critical for the nitritation and denitrification of landfill leachate. In order to realize nitritation, enrichment of ammonia oxidizing bacteria (AOB) and inhibition on nitrite oxidizing bacteria (NOB) should be realized in reactor. Thus, controlling strategies such as high temperature, low dissolved oxygen (DO) and free ammonia (FA) inhibition have been commonly reported (Peng and Zhu, 2006). Though the growth rate of AOB is higher than that of NOB at temperature higher than 25 °C (Hellinga et al., 1998), it is not suitable for practical operation due to extra energy needed. DO lower than 1.0 mg/L was frequently reported for achieving nitritation of landfill leachate (Canziani et al., 2006; Kulikowska and Bernat, 2013; Chen et al., 2016), while
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strict operation should be considered for low DO level and nitritation can be easily destroyed when DO is out of control. Since oxygen plays the role of electron acceptor in ammonia oxidation, low DO levels may also lead to a low ammonium removal rate as well as low biomass yield (Wang and Yang, 2004). It was reported that inhibition on AOB and NOB appeared with FA level of 10–150 mg/L and 0.1–1.0 mg/L, respectively (Anthonisen et al., 1976). As ammonium in landfill leachate is typically high, FA inhibition is another common approach for nitritation of landfill leachate (Nhat et al., 2017; Sun et al., 2014; Zhang et al., 2015). However, in many cases, FA concentration is hard to keep stable because of its gradual decrease by ammonia removal during aeration process, leading to final failure of inhibition on NOB (Wei et al., 2014) and damage of nitrite accumulation. That is to say, stable nitrite accumulation is still an obstacle by strategies above. Compared to high temperature or low DO, FA inhibition may be more available since there is no extra energy needed or substrate limit for AOB (oxygen or ammonia). However, how to maintain stable FA levels in the reactor is still a challenge. Zeolite is a superior adsorbent for ammonium, which was also reported to be biofilm carrier for enrichment of AOB (Li et al., 2013). During the ammonium adsorption, adsorption equilibrium will eventually appear and portion of ammonium remains in liquid phase (Chen et al., 2018a). Based on this, the application of zeolite for biofilm carrier has the potential for keeping appropriate FA levels and stable nitritation can be obtained by persistent FA inhibition on NOB in during high-strength ammonium wastewater treatment. This was verified by Yang et al. (2017) who found that zeolite biological aeration filter (ZBAF) could successfully realize efficient nitritaion with stable nitrite accumulation for high concentration ammonium wastewater. This finding showed ZBAF might be feasible for nitritation and denitrification of landfill leachate. However, relevant report about ZBAF application for landfill leachate treatment is scarcely reported and whether this reactor is suitable for nitritation and denitrification of landfill leachate is still unclear. In a municipal solid waste landfill plant in Suixi, Guangdong province, China, landfill leachate was disposed by two-stage anoxic-oxic process for nitrogen removal pretreatment (Fig. S1), in which large amount of glucose was added for denitrification (Table S1). Due to the high cost for carbon source, the plant looked forward to cost-effective nitrogen pretreatment process, such as nitritation and denitrification. Thus, a pilot-scale predenitrification and ZBAF process (about 1 m3/d) was established on site and carried out for landfill leachate nitrogen and organic matter removal. During this pilot study, removal of organic matter and nitrogen were investigated. In addition, nitritation performance of ZBAF was estimated and potential nitritation mechanism of landfill leachate by ZBAF was also discussed. Moreover, variations of microbial community structure of biomass on the zeolite were also analyzed to verify nitritation performance. This study intended to estimate the feasibility of this process for nitritation and denitrification of low C/N landfill leachate.
2. Materials and methods 2.1. The pilot apparatus for landfill leachate treatment The scheme of the pilot-scale apparatus used in this study were shown in Fig. 1, which contained influent tank (PE, V = 2 m3), anoxic tank (PE, V = 5 m3), settling tank (PE, V = 2 m3), buffer tank (PE, V = 1 m3), ZBAF (anticorrosive carbon steel, u0.95 4.5 m) and effluent tank (PE, V = 2 m3), respectively. Both the influent tank and anoxic tank were equipped with mechanical mixer for stirring. The ZBAF was filled with 1–2 mm natural zeolite as biofilm carrier and
the height of zeolite carrier was about 2.5 m. Air was supplied to ZBAF for aeration. Landfill leachate was firstly mixed with 1000 mg/L glucose batch by batch in the influent tank. Then it was transported to the anoxic tank mixed with ZBAF effluent reflux by stirring for denitrification. The effluent of ZBAF was controlled by adjusting valves for desired reflux ratio. After denitrification, the influent was settled in the settling tank and flowed to the buffer tank by gravity. Then it was pumped into ZBAF for nitritation and effluent flowed to the effluent tank. During the pilot study, the influent flow rate of landfill leachate and ZBAF were all controlled by adjusting the rotor flow meter. DO in ZBAF was controlled in the range of 6.0–7.4 mg/L and temperature was kept at 28.0–33.0 °C. 2.2. Landfill leachate and seeding sludge The landfill leachate used for this study was obtained from a leachate reservoir in municipal solid waste landfill plant in Suixi, Guangdong province, China. Its characteristics were listed in Table 1. According to Table 1, it was certain that total nitrogen (TN) mainly presented as ammonium in this landfill leachate. In this treatment plant, landfill leachate was treated by a twostage anoxic and oxic process for nitrogen removal with hydraulic retention time (HRT) of about 30 days. Seeding sludge was picked from one oxic tank (500 m3/d) of the local landfill leachate treatment plant for start-up of anoxic tank and ZBAF. The mixed liquor suspended solid (MLSS) of seeding sludge was about 6000– 7000 mg/L. 2.3. Experimental operation and procedure ZBAF was first run with batch influent and continuous aeration to cultivate biofilm for 15 days. After the start-up of ZBAF, the pilot study was carried out according to Table 2. The landfill leachate inflow rate increased stepwise to 30, 35, 38 and 40 L/h, respectively, and reflux ratio was reduced to increase HRT (or reduce upflow rate) of ZBAF for releasing biological inhibition by high concentration ammonium. Based on the operational condition for twostage anoxic-oxic process in the landfill leachate treatment plant (Fig. S1), in order to obtain higher nitrogen removal efficiency, 1000 mg/L glucose was added into the influent tank to increase C/N ratio for denitrification. During this study, the removal of organic matter and nitrogen pollutants by nitritation and denitrification and ZBAF nitritation performance and potential mechanism were investigated. 2.4. Chemical analysis and calculations Chemical oxygen demand (CODCr), NH+4-N, nitrite (NO 2 -N) and nitrate (NO 3 -N) were respectively detected every day by potassium dichromate reflux method, Nessler’s reagent spectrophotometer method, N-(1-Naphthalene)-Ethylenediamine dihydrocholride spectrophotometer method, ultraviolet spectrophotometer according to Chinese standard examination of water and wastewater, respectively(China, 2002). All CODCr data were corrected according to the fact that nitrite nearly exerts a CODCr of 1.1 mg O2/mg NO 2 -N (Li et al., 2014). Since organic nitrogen in landfill leachate was very low, sum of NH+4-N, NO 2 -N and NO 3 -N was chosen to represent TN in samples. The temperature and DO were both measured by a digital DO meter (HQ30d, HACH, USA) and pH of every sample was measured by a pH meter (PHS3C, INESA Scientific Instrument Co. Ltd, China). The ZBAF packing ammonium loading rate (ALR), nitrite production rate (NPR), nitrite accumulation ratio (NAR) were calculated according to Eqs. (1), (2) and (3), respectively. FA was estimated by Eq. (4) reported by Anthonisen et al. (1976).
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Fig. 1. The schematic diagram of pilot study for landfill leachate treatment: (a) Schematic diagram, (b) Site picture.
Table 1 Characteristics of landfill leachate.
*
Content
pH
CODCr (mg/L)
NH+4-N (mg/L)
NO— 2 N (mg/L)
NO— 3 N (mg/L)
TN (mg/L)
Alkalinity (mgCaCO3/L)
Conductivity (mS/cm)
Range
8.7–9.0
6140–6725
2250–3341
ND*
5.8–11.9
2345–3362
10,500–12,400
28.3–31.4
ND means no detected.
Table 2 Operational scheme of the pilot study. Day
1–4 5–9 10–13 14–16 17–24 25 26–28 29–32
Influent flow rate (L/h)
30 35 35 35 38 40 40 40
Reflux ratio
10 8.43 9 7 6.37 6 5 4
ZBAF Inflow rate (L/h)
HRT (h)
Upflow rate (m/h)
DO (mg/L)
T (oC)
330 330 350 280 280 280 240 200
5.36 5.36 5.06 6.32 6.32 6.32 7.38 8.85
0.466 0.466 0.494 0.396 0.396 0.396 0.339 0.282
6.0–7.4
28.0–33.0
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NHþ4 N effluent NHþ4 N influent ALR kg NHþ4 N=ðm3 dayÞ ¼ 24 HRT 1000 ð1Þ
NO2 Neffluent NO2 Ninfluent NPR kg NO2 N=ðm3 dayÞ ¼ 24 HRT 1000 ð2Þ
NARð%Þ ¼
NO2
FAðmg=LÞ ¼
NO2 Neffluent 100 Neffluent þ NO3 effluent
17 NHþ4 N 10pH h i 100% 14 exp 6334 þ 10pH
ð3Þ
ð4Þ
273þT
Mass balance analysis was carried out based on the input and output pollutant of each operational unit. The concentration varia tion of each pollutant DX (CODCr, NH+4-N, NO 2 -N or NO3 -N) in each operational unit was calculated according to Eq. (5) as followed.
DX ¼
X output X input Q out
ð5Þ
where Xinput and Xoutput was the total mass amount pollutant input or output per unit time of each operational unit, mg/h; Qout was the output flow rate of each operational unit, L/h. The plus or minus sign of DX represented that pollutant concentration increased or decreased in that operational unit. 2.5. Metagenomic DNA extraction and high-throughput sequencing analysis 2.5.1. Extraction of DNA from microbiological samples Seeding sludge from the oxic tank of local landfill leachate treatment plant was picked at the beginning of pilot study (named O1) and at day 30 (named O2), and bio-zeolite sample was also collected at day 10, 20, 25 and 30, named as Z1, Z2, Z3 and Z4, respectively. All microbiological samples were washed as per protocol described earlier (Chen et al., 2016) and extracted for total genomic DNA using a Bacterial DNA Isolation Kit (50, D3350-01, OMEGA, USA) according to the manufacturer’s instructions. The DNA samples concentration and quantity were all detected by ultraviolet and visible spectrophotometer at 260 and 280 nm, and Agarose Gel Electrophoreses, respectively. 2.5.2. Amplicon generation and purification The amplicon generation of 16S rRNA was performed using the specific primer 319F-806R for 16S V4 with the barcode (Yang et al., 2017) on Illumina Miseq PE250 platform. The PCR reaction was carried out with a total volume of 50 lL and 30 ng genome DNA, 4 lL of PCR Prime Cocktail, 25 lL of Phusion High-Fidelity PCR Master Mix (New England Biolans) was used. The detailed operation of PCR included initial denaturation at 98 °C for 3 min, 30 cycles of denaturation at 72 °C for 45 s; extension at 72 °C for 7 min; and finally holding at 4 °C. The PCR productions were purified with AmpureXPbeads (AGENCORT) for removing the unspecific products. 2.5.3. Statistical analysis In order to obtain more accurate and reliable results in subsequent bioinformatics analysis, the raw data was pre-processed to get clean data by in-house procedure as following: (1) Truncation of sequence reads nor having an average quality of 20 over a 30 bp sliding window based on the phred algorithm, and trimmed reads having less than 75% of their original length, as well as its paired read, will be removed; (2) Removal of reads contaminated by adapter (default parameter: 15 bases overlapped by reads and
adapter with maximal 3 bases mismatch allowed); (3) Removal of reads with ambiguous base (N base), and its paired reads; (4) Removal of reads with low complexity (default: reads with 10 consecutive same base). For pooling library with barcode samples mixed, the clean reads were assigned to corresponding samples by allowing 0 base mismatch to barcode sequences with inhouse scripts. The reads with sequencing adapter, N base, poly base, low quality etc., were filtered out with default parameters aforementioned (Table S2). If the two paired-end reads overlapped, the concensus sequence was generated by FLASH (Fast Length Adjustment of Short reads, v 1.2.11), and the detailed method is as follows: (1) Minimal overlapped length: 15 bp; (2) Mismatching ratio of overlapped region: 0.1; (3) Removal of paired-end reads without overlaps. The high quality paired-end reads were combined to tags based on overlaps. 174,873 tags were obtained in total with 29,145 tags per sample on average (Table S3), and the average length was 253 bp. The tags obtained were clustered to OTU (Operational Taxonomic Unit) by script of software USEARCH (v7.0.1090) with a 97% similarity threshold by using UPARSE, and the OTU unique representative sequences were obtained. Chimeras were filtered out by using UCHIME (v4.2.40). All tags were mapped to each OTU representative sequencing using USEARCH GLOBAL, then the tags number of each OTU in each sample was summarized to OTU abundance table. Chao, Ace, Shannon index and Simpson index was calculated for alpha diversity analysis. The Illumina high-throughput sequencing methods and data analysis were under the consideration of quality assurance and quality control. QIIME was used for analyzing the clustering of taxonomic based on OTUs. 3. Results and discussion 3.1. Performance of the pilot study Fig. 2 showed the overall performance of the pilot study for the treatment of landfill leachate. According to Fig. 2(a), influent and effluent CODCr of the pilot apparatus ranged from 6200 to 6800 mg/L and 2600 to 3300 mg/L, respectively, leading to an overall average CODCr removal efficiency of 53.2 ± 3.0%. In Fig. 2(b), from day 10 to day 24, effluent NH+4-N stayed stable at 183.7 ± 23.2 mg/L with an approximate removal efficiency of 93.5 ± 1.2%. When inflow rate increased to 40L/h, however, effluent NH+4-N increased from 200 to 300 mg/L and its removal efficiency also decreased to about 90.0%. It could be seen in Fig. 2(c) that TN removal efficiency gradually decreased along with increase of inflow rate. With inflow rate of 40 L/h, average TN removal efficiency of the pilot apparatus was about 66.0 ± 6.3% under an average ALR of 1.93 ± 0.26 kg NH+4-N m3 day1 on ZBAF. All in all, it was clear that CODCr, NH+4-N and TN were significantly removed from landfill leachate by the pilot apparatus. 3.2. Pollutants removal in different units The detailed variations of CODCr, NH+4-N, NO 2 -N, NO3 -N and pH in anoxic tank, buffer tank and ZBAF were listed in Fig. 3(a), (b), (c), (d), (e), respectively. Compared to influent for this study, CODCr ranged at 3000–3500 mg/L in anoxic tank and then slightly declined in buffer tank and ZBAF. According to Fig. 3(b) and (c), compared to anoxic tank and buffer tank, average effluent NH+4-N and NO 2 -N of ZBAF was 180.0 ± 62.1 and 499.4 ± 189.0 mg/L, respectively, showing remarkable NH+4-N decrease and NO 2 -N accumulation in ZBAF. In Fig. 3(d), average effluent NO 3 -N of anoxic tank, buffer tank and ZBAF was only 15.2 ± 5.8, 14.4 ± 6.4 and 31.9 ± 7.4 mg/L during the whole pilot study, respectively.
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(DCODCr = 430.0 mg/L) and TN (DTN = 245.2 mg/L). It should be pointed out that majority of removed nitrogen in anoxic tank was nitrite (DNO 2 -N = 208.5 mg/L), which was offered by reflux from ZBAF. These data revealed denitrification via nitrite pathway occurred in the anoxic tank indeed. Besides, a portion of CODCr and TN were also removed in buffer tank, which revealed that denitrification should also happen in buffer tank. In ZBAF, ammonium was apparently removed withDNH+4-N as 311.0 mg/L and nitrite accumulated dramatically withDNO 2 -N as 444.0 mg/L. On the contrary, Nitrate was reduced in ZBAF(DNO 3 -N = 22.8 mg/L). As ZABF was a kind of biofilm reactor, anoxic microenvironment might be easily created and then denitrification could happen with a few organic matter (Ryu et al., 2008). Since CODCr also decreased in ZBAF (DCODCr = 61.8 mg/L), it could be speculated that nitrate denitrification possibly occurred. The concentration difference between NO 2 -N and NO3 -N in the ZBAF effluent meant that NOB was deeply inhibited and excellent nitrite accumulation was achieved in ZBAF, indicating this reactor played a key role in achieving stable nitritation of the landfill leachate. In addition, TN also increased in ZBAF on day 20 (DTN = 110.2 mg/L), possibly resulting from the ammonium release of zeolite fillings. This finding meant that zeolite fillings might show an effect on nitrogen concentration balance in ZBAF by ammonium release or adsorption with influent ammonium concentration fluctuations (Yang et al., 2017). 3.3. Nitritation performance and mechanism of ZBAF
Fig. 2. The overall performance of pilot study in treating landfill leachate: (a) CODCr, (b) NH+4-N, (c) TN.
Fig. 3(e) showed pH value in effluent of anoxic tank, buffer tank and ZBAF was 9.02 ± 0.05, 9.20 ± 0.07 and 8.61 ± 0.23, respectively. In order to better understand the pollutants removal pathway in this study, analysis data on day 20 was picked as an example for mass balance analysis (Fig. 4). After mass balance calculation, pollutant concentration variations DX in each operational unit were listed in Table 3. According to mass balance analysis results, the anoxic tank was the main reactor for removal of organic matter
In order to estimate nitritation performance of ZBAF, ALR and NPR was calculated and shown in Fig. 5(a). Combined with Fig. 3, from day 1 to day 9, even though influent ALR increased simultaneously from 1.520 to 2.018 kg NH+4-N m3 day1, NPR of ZBAF kept 3 in a rising trend from 0.468 to 1.354 kg NO day1. When 2 -N m ALR increased to 2.868 ± 0.095 kg NH+4-N m3 day1 during day 10 13, significant drop of NPR happened, with lowest NPR of 3 0.691 kg NO day1. With influent upflow rate of 2 -N m 0.396 m/h during day 14–24, highest NRP appeared as about 3 1.686 kg NO day1. However, when influent inflow rate 2 -N m increased to 40 L/h after day 24, NRP decreased again even though influent ALR decreased to about 1.927 ± 0.261 kg NH+4-N m3 day1. As aforementioned, ZBAF showed efficient nitritation in this study. According to Fig. 5(b), FA in ZBAF during the whole study was typically high meanwhile NAR was basically higher than 90.0% in most of cases. Even though influent NH+4-N of ZBAF varied from 293.4 to 687.8 mg/L, effluent NH+4-N of ZBAF always kept in the range of 81.8–306.0 mg/L, which should be attributed to the frequently automatic shift of adsorption equilibrium by zeolite in ZBAF (Yang et al., 2017). Due to this phenomenon, ZBAF could maintain an appropriate FA range and also showed buffer function in spite of different influent ammonium concentration. In the literature, FA concentration specifically differed for sustainable landfill leachate nitritation and deep inhibition on NOB. Zhang et al. (2015) found that nitrite accumulation rate remained high with FA from 5.30 to 48.67 mg/L in landfill leachate treatment, while maximum FA concentration as high as 506 mg/L was also reported for nitritation of old landfill leachate (Nhat et al., 2017). In this study, influent NH+4-N for ZBAF gradually increased from day 1 to day 9 with FA range of 22.83–50.08 mg/L. During this period, NPR kept rising 3 and reached 1.354 kg NO day1 at day 9, indicating that 2 -N m this FA range showed distinct inhibition on NOB but not AOB in ZBAF. However, with upflow rate increased to 0.494 m/h during day 10–13, significant drop of NPR appeared as FA increased to 64.72–92.34 mg/L, which provided evidence that AOB was also inhibited. By decreasing upflow rate increased to 0.396 m/h, this situation changed quickly with an average NPR of 3 1.400 ± 0.231 kg NO day1 even though landfill leachate 2 -N m
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Fig. 3. Typical profiles of CODCr, nitrogen concentration and pH in different units: (a) CODCr, (b) NH+4-N, (c) NO 2 -N, (d) NO3 -N, (e) pH.
inflow rate increased to 38 L/h. These results further demonstrated that FA inhibition on AOB in ZBAF might be reversible. When inflow rate increased to 40 L/h, NPR dropped again with obvious effluent NO 2 -N decrease, which might be attributed to severe FA inhibition on AOB by range of 63.20–93.81 mg/L. Based on results above, it could be concluded that influent flow rate should decide nitritation performance of ZBAF. The total vessel volume of the whole pilot apparatus was about 9.8 m3, including anxic tank, settling tankd, buffer tank and ZBAF packings. Therefore, in order to obtain desired nitritation performance, total HRT of the process for landfill leachate should not less than 10.2 days. Since the oxygen affinity constant of AOB and NOB was 0.3– 0.5 mg/L and 0.7–1.8 mg/L, respectively (Guisasola et al., 2005), low DO concentration was chosen for nitritation. Nevertheless, DO in ZBAF was typically as high as 6.0–7.4 mg/L in this pilot study. Thus, DO could not be the main factors for inhibiting NOB and realizing stable nitritation. It was reported that growth rate of AOB was higher than that of NOB with temperature higher than 20 °C (Hellinga et al., 1998). The temperature in ZBAF varied from 28.0 to 33.0 °C, suggesting that effect of temperature on promoting nitritation should be taken into consideration. According to Fig. 3 (e), pH in ZBAF maintained at the range of 8.12–9.01 during the whole study, which could result in a higher FA value compared to the situation that pH less than 8.0 according to Eq. (4). Based on the temperature and pH value, FA of ZBAF kept in 22.84– 93.81 mg/L. This FA was higher than FA of former study ranging in 17.35–40.87 mg/L for determining excellent nitritation performance in ZBAF (Yang et al., 2017). Therefore, FA inhibition should be the main reason explaining stable and efficient nitritation in ZBAF for this pilot study. Besides, high DO levels presented no damage on nitrite accumulation in ZBAF. Instead, this DO level could offer more oxygen for metabolism of AOB to promote nitritation performance (Regmi et al., 2014). This finding showed the advantage of ZBAF for nitrtitaion of landfill leachate compared to other methods and further revealed the feasibility and superiority of ZBAF.
3.4. Microbial community taxonomic analysis The microbial community variations of biofilm on zeolite carriers compared to inoculated sludge were investigated by highthroughput sequencing technology. Table 4 listed the values of effective reads, OTUs, Chao and Shannon index of all biological samples. With the operation of the pilot-scale process, OTUs, Chao and Shannon index all increased as running time increased, showing gradual microbial diversity improvement in ZBAF. Compared O1 with O2, these results demonstrated that microbial communities in ZBAF changed. According to NMDS analysis results (Fig. 6), obvious distance between zeolite biofilm samples and sludge samples indicated the difference of community structure between ZBAF and oxic tank. In Fig. 7 taxonomic classifications of all samples were presented at phylum and class level. For the inoculated sludge (O1), three most abundant bacteria at phyla level were Bacteroidetes (46.37%), Proteobacteria (24.49%) and Thermi (8.09%), and then shifted slightly with Bacteroidetes (56.00%), Proteobacteria (23.37%) and Thermi (13.57%) after about a month operation of oxic tank. Due to the continuous operation under inhibition of FA in ZBAF, remarkable microbial community variation happened in ZBAF compared to the oxic tank, with Bacteroidetes (43.92%, 44.38%, 44.52%, 46.52%), Proteobacteria (44.38%, 43.09%, 44.06%, 41.28%) and Thermi (3.00%, 3.44%, 2.98%, 2.47%) at day 10, 20, 25, 30, respectively. The noteworthy relative abundance of Proteobacteria in ZBAF showed dominance of this phylum, which was similar to results that both of efficient NH+4-N removal and nitrite accumulation appeared with richness of Proteobacteria (Wang et al., 2014). In addition, Betaproteobacteria at class level of all ZBAF samples were higher than that of the sludge samples, which further explained the reason for nitrite accumulation in ZBAF (Gao et al., 2011; Chen et al., 2016). Besides, class Nitrospira were all detected in O1 and O2 with relative abundance of 4.07% and 1.29%, while none of this class was detected in each of zeolite biofilm samples, which revealed the washout of NOB in ZBAF.
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Fig. 4. Mass balance analysis of CODCr and nitrogen based on data of day 20.
Table 3 Pollutant concentration variations in different unit on day 20 after mass balance analysis Unit: mg/L. Unit
Anoxic tank
Buffer tank
ZBAF
DCODCr DNH+4-N DNO 2 -N DNO 3 -N DTN
430.0 19.1 208.5 17.6 245.2
107.5 5.0 135.0 2.1 142.1
61.8 311 444 22.8 110.2
In the literature, accumulation of AOB and washout of NOB were commonly reported in study of nitritation (Daims et al., 2015; Fitzgerald et al., 2015). For ZBAF, Yang et al. (2017) also found that Nitrosomonas was typically dominant in ZBAF for high
concentration ammonium wastewater treatment. In this pilot study, the relative abundance of Nitrosomonas at genus level in ZBAF was found to be 22.54%, 21.72%, 21.78% and 20.15% at day 10, 20, 25, 30, respectively, while genus Nitrospira was undetectable (Table 5). These findings definitely verified the dominance of AOB and washout of NOB by severe FA inhibition in ZBAF with FA as high as 22.83–93.81 mg/L in the pilot study. In other words, ZBAF was proved to be feasible for stably efficient nitritation of landfill leachate.
3.5. Significance of this study and issue to be addressed Based on the nitritation performance of ZBAF, this pilot study presented with pre-denitrification and post-nitritation was suc-
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Fig. 5. ALR, NPR, NAR and FA in ZBAF during the pilot study: (a) ALR and NPR, (b) NAR and FA.
Table 4 The numbers of reads and alpha diversity results.a Sample ID Reads Alpha diversity
a b c d
Raw Effective OTUsb Chaoc Shannond
O1
O2
Z1
Z2
Z3
Z4
30,076 28,956 476 502 3.732
30,650 29,573 388 439 2.864
29,990 28,937 429 461 3.519
30,041 29,082 432 466 3.585
30,307 29,344 458 520 3.602
30,413 29,482 456 535 3.701
Similarity level = 0.97. OTUs: operational taxonomic units (calculated at the 3% distance limit). Chao is a kind of alpha diversity index reflecting microbial community species richness of sample. A higher number means a higher richness. Shannon is a kind of alpha diversity reflecting microbial community species diversity of sample. A higher number represents more diversity.
Fig. 6. NMDS analysis results of different samples.
cessfully applied for organic matter and nitrogen removal from landfill leachate. During this study, average CODCr, NH+4-N and TN removal efficiency was 53.2 ± 3.0%, 93.5 ± 2.4% and 74.7 ± 9.4% 3 respectively with an average NPR of 1.387 kg NO day1. 2 -N m The operational cost for this landfill leachate was mainly decided by energy needed for aeration and dosage of extra carbon source. During the whole study, although extra carbon source (1000 mg/ L glucose, about 0.62 US dollar/m3) was added in influent, nitrite could not be removed totally in anoxic tank or buffer tank (Fig. 3 (c)) so that TN removal was not thorough. As the theoretical C/N ratio for nitrite denitrification was 1.78 (Chen et al., 2018b), higher
TN removal efficiency could be realized by increasing glucose dosage in influent. According to data in Table S1, TN removal efficiency of two-stageanoxic-oxic process in the plant was about 78.1% with 6000 mg/L glucose (about 3.72 US dollar/m3) dosed, which was only 3.4% higher than that of presented process. That was to say, leaving out energy consumed for aeration, approximate 5000 mg/L glucose (about 3.10 US dollar/m3) could be saved to achieve almost similar TN removal performance. The plant could save about 310 US dollar/day if the presented process based on ZBAF was applied for nitrogen removal pretreatment of low C/N landfill leachate. Currently, most of pretreatment process for low C/N ratio landfill leachate in China is still the traditional two-stage anoxic/oxic process, in which ammonium is converted by complete nitrification and nitrogen is removed by nitrate denitrification. In this case, many landfill leachate treatment plants are facing the situation that operational cost for landfill leachate pretreatment stays at a high level because of the extra dosage of carbon source. However, this situation can definitely change if the presented process based on ZBAF reported in this study is applied for landfill leachate pretreatment, which shows potential application prospect in saving operational cost by realizing stable nitritation and denitrification. In addition, it should be noted that both of NH+4-N and NO 2 -N existed in the effluent in a certain concentration range. From this point of view, ANAMMOX might be possible for subsequent nitrogen removal of effluent if the ratio of NH+4-N and NO 2 -N in effluent could be controlled by adjusting operational parameters of ZBAF. That is to say, a promising nitrogen removal process based on ZBAF and ANAMMOX for low C/N landfill leachate is potentially possible. Besides, the effect of potential ammonium release from zeolite in ZBAF still needed investigation in the future.
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Fig. 7. Taxonomy classification of bacterial diversity at Phylum and Class level of seed sludge and ZBAF: (a) Phylum level, (b) Class level.
Table 5 Comparison of relative abundance of AOB and NOB for different samples.
*
Sample
O1
O2
Z1
Z2
Z3
Z4
Nitrosomonas Nitrospira
3.21 3.75
2.24 1.23
22.54 ND*
21.72 ND
21.78 ND
20.15 ND
ND means no bacterium at genus level was detected.
4. Conclusions The pilot scale pre-denitrification and ZBAF process succeeded in achieving stable nitritation and denitrification for low C/N landfill leachate. During the whole operation, removal efficiency of CODCr, NH+4-N and TN of pilot scale process was 53.2 ± 3.0%, 93.5 ± 2.4% and 74.7 ± 9.4%, respectively. In this process, ZBAF was the key for stable nitrite accumulation with NAR higher than 90.0%. Based on the zeolite fillings, ZBAF showed excellent buffer function with frequent variation of influent NH+4-N concentration and maintained an appropriate FA range for inhibition on NOB, which was the main factor for efficient nitritation. Highthroughput sequencing analysis results further revealed enrichment of AOB and elimination of NOB definitely happened in ZBAF. The nitrogen removal pretreatment process based on ZBAF was highly promising for low C/N landfill leachate treatment. Acknowledgement This work was supported by the Specialized Applied Science and Technology Research, Development and Major Transformation Project of Guangdong Province in 2017 (2017B020236004). Authors are also thankful to Weilong Li and the staffs of Suixi landfill leachate treatment plant for their kindly helps. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.09.020. References Anthonisen, A.C., Loehr, R.C., Prakasam, T.B.S., Srinath, E.G., 1976. Inhibition of nitrification by ammonia and nitrous acid. Water Pollut. Control Federat. 48 (5), 835–852. Canziani, R., Emondi, V., Garavaglia, M., Malpei, F., Pasinetti, E., Buttiglieri, G., 2006. Effect of oxygen concentration on biological nitrification and microbial kinetics in a cross-flow membrane bioreactor (MBR) and moving-bed biofilm reactor (MBBR) treating old landfill leachate. J. Membr. Sci. 286 (1–2), 202–212.
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