Simultaneous partial nitrification, anammox and denitrification (SNAD) process for nitrogen and refractory organic compounds removal from mature landfill leachate: Performance and metagenome-based microbial ecology

Bioresource Technology 294 (2019) 122166

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Simultaneous partial nitrification, anammox and denitrification (SNAD) process for nitrogen and refractory organic compounds removal from mature landfill leachate: Performance and metagenome-based microbial ecology

T

Yingmu Wanga,b, Ziyuan Lina,b, Lei Hea,b, Wei Huanga,b, Jian Zhoua,b, , Qiang Hea,b ⁎

a b

Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, China National Centre for International Research of Low-Carbon and Green Buildings, Chongqing University, Chongqing 400045, China

GRAPHICAL ABSTRACT

ARTICLE INFO

ABSTRACT

Keywords: Mature landfill leachate SNAD Nitrogen removal Refractory organics removal Metagenome analysis

In this study, a simultaneous partial nitrification, Anammox and denitrification (SNAD) bioreactor was constructed for mature landfill leachate treatment, which exhibited favorable NH4+-N (98.9–99.9%), TN (90.7–94.9%) and bio-refractory organic compounds (46.2–67.7%) removal efficiencies. Stoichiometric analysis demonstrated that the synergy of ammonium-oxidizing bacteria and Anammox bacteria dominated TN removal (96.1–97.2%). NO3−-N produced in Anammox could be further reduced through (partial) denitrification and dissimilatory nitrate reduction to ammonium (DNRA). The results highlighted that humic-like and their intermediates might serve as the electron donor for these (partial) denitrifiers and DNRA bacteria to remove NO3−-N, and could be effectively removed from mature landfill leachate in SNAD bioreactor. Metagenomic characterization further demonstrated that phyla Chloroflexi, Chlorobi and genera Nitrosomonas, Ignavibacterium and Aminiphilus might be responsible for such humic-like degradation. Overall, this work offers new insights into the metagenome-based bioinformatic roles for the previously understudied microorganisms in SNAD bioreactor for mature landfill leachate treatment.

⁎ Corresponding author at: Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, China. E-mail address: [email protected] (J. Zhou).

https://doi.org/10.1016/j.biortech.2019.122166 Received 6 August 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 20 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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1. Introduction

and efficient genetic tool for investigating the functional organisms and their potential for nitrogen and carbon cycling (Fang et al., 2013; Speth et al., 2016). Therefore, to gain comprehensive insights into the ecosystem functioning concerning the intrinsical nitrogen and carbon metabolisms in SNAD system for mature landfill leachate treatment, a metagenome pipeline was performed to gain the functional pathways and ecological model based on retrieved near-complete genome sequences. In this study, an SNAD bioreactor fed with mature landfill leachate was established based on a previously reported PNA process fed with synthetic ammonia-rich wastewater (Zhang et al., 2014). The main aims of work were to (1) evaluate the performance for simultaneous removals of nitrogen and refractory organic matters from mature landfill leachate in SNAD system; (2) investigate the conversion pathways of refractory organic compounds from mature landfill leachate; (3) elucidate the linkage among microbial ecology, functional microorganisms, and biochemical reactions concerning nitrogen and refractory organic compounds transformations based on metagenome analysis. The results of this study provide a system-wide and comprehensive overview of the microbial community and ecosystem functioning of SNAD process for the treatment of mature landfill leachate and similar industrial wastewater.

Currently, sanitary landfill remains the most common management strategy for the municipal solid wastes (MSW) disposal owing to its economic advantages, especially in developing countries (Hoornweg and Bhada-Tata, 2012; Pandey and Shukla, 2019). In China, there were 654 MSW landfills operating in 2017, in which 120.37 billion tons of the total disposal MSW were received, accounting for 57.2% of MSW disposal. However, landfill leachate, a by-product of landfills, contains highly concentrated ammonium (NH4+-N), organic wastes and toxic materials (Xiong et al., 2018), which is a potential anthropogenic contamination source of the received waterbody. Therefore, there is an urgent need to reduce nitrogen and organic inputs from MSW landfills for aquatic ecosystem maintenance and recovery. Nitrification/denitrification process has been demonstrated to be feasible for simultaneous nitrogen and COD removals from immature landfill leachate (Chen et al., 2016; Zhu et al., 2013). However, as mature landfill leachate (under landfilling for more than five years) contains lower biodegradable organic matters (Wang et al., 2016), it is difficult to facilitate denitrification process for simultaneous nitrogen and COD removals from mature landfill leachate (Cassano et al., 2011; Zhang et al., 2017). In addition, external organic carbon source dosing for denitrification is not recommended owing to its uneconomic disadvantages. Recently, the coupling of partial nitrification (PN) and anaerobic ammonium oxidation (Anammox) has been demonstrated to be suitable for nitrogen removal from mature landfill leachate (Wen et al., 2016; Xie et al., 2013). The integration of PN/Anammox (PNA), could be achieved in two-stage reactors such as SHARON-Anammox process, and in single-stage reactors such as completely autotrophic nitrogen removal over nitrite (CANON) process (Wen et al., 2016). When compared with traditional nitrification/denitrification, PNA process allows 63% lower aeration demand, no external carbon dosing, less greenhouse gas emission, and lower sludge yields (Bagchi et al., 2012; Speth et al., 2016). Theoretically, < 88.8% of total nitrogen (TN) could be removed by PNA process, because about 11.2% of nitrate (NO3−-N) was formed in the effluent (Bagchi et al., 2012). Additionally, PNA process do not focus on refractory organic compounds elimination from mature landfill leachate. Therefore, any effort for the enhancement of TN and COD removal effectiveness for mature landfill leachate treatment should be addressed to meet the stringent TN and COD discharge standard. Alternatively, simultaneous partial nitrification, Anammox and denitrification (SNAD) process could enhance simultaneous TN and COD elimination from landfill leachate (Wang et al., 2018). In SNAD system, the produced NO3−-N during Anammox process could be mitigated by (partial) denitrifiers that catalyze the conversion of NO3−-N back to NO2−-N and/or N2 (Speth et al., 2016; Zheng et al., 2019b). Besides, the refractory organic matters in mature landfill leachate might be removed by certain heterotrophs coexisted in Anammox-based bioreactors (Li et al., 2018b; Wang et al., 2018). Compared with PNA process, SNAD process emphasize the effect of denitrification coupling with PN/Anammox processes on simultaneous N and organic compounds removal as well as better TN removal efficiency. However, the transformation pathways concerning simultaneous nitrogen and refractory organic compounds in mature landfill leachate, and their bioinformatic microbial ecology as well as the metabolic potential of SNAD bioreactor, have not been investigated to date. Recently, qPCR and 16S rRNA sequencing techniques have provided microbial insights into the linkage between phylogenetic biodiversity and certain organisms concerning several specific biochemical reactions (e.g., Anammox) within mature landfill leachate in SNAD system (Connan et al., 2018; Wang et al., 2018; Zheng et al., 2019a). However, little bioinformation is known about the linkage among the diversity of functional genes, metabolic potential, and ecosystem functioning in SNAD system for mature landfill leachate treatment only by 16S rRNA gene inventories. Metagenome analysis has proven to be an emerging

2. Materials and methods 2.1. Mature landfill leachate In this work, the mature landfill leachate was collected from Changshengqiao MSW landfill (Chongqing, China). The concentrations of NH4+-N and COD in raw leachate were 2004.8 ± 14.7 and 1026.8 ± 14.2 mg L−1, respectively. The values of BOD5 in the raw leachate were only 16.7 ± 5.4 mg L−1, and the average BOD5/COD ratio of mature landfill leachate was 1.6%, featuring notably poor in biodegradation of organic compounds. This indicated that the concentration of bio-refractory organic compounds was close to COD value in the raw leachate. The compositions of the collected mature landfill leachate in this study were detailed in Table 1. 2.2. Experimental setup and procedure In this work, a single-stage sequencing batch biofilm reactor (SBBR) fed with mature landfill leachate was established. The SBBR was inoculated with biofilm biomass from a bench-scale PNA bioreactor enriched with Anammox bacteria (AMX) and ammonium-oxidizing bacteria (AOB). The SBBR in this work was composed of polymethyl methacrylate, with an effective volume capacity of 10.0 L. Semi-soft fiber was used as biofilm carrier, and the packing fraction of the bioreactor was 50% (V/V). The ambient temperature of the reactor was controlled at 30.0 ± 1.0 °C by using a thermostatic chamber (Boxun, SPX-150B-Z, China). Intermittent aeration was adopted to create alternate micro-aerobic and anoxic conditions. The intermittent aeration cycle of the SBBR was 8 h, consisting of aeration period (4 h) and nonaeration period (4 h). The dissolved oxygen (DO) concentration was maintained at 2.2–2.4 mg L−1 at the aeration period, which was similar to our previous work (Wang et al., 2018; Wen et al., 2016). Feeding Table 1 Characteristics of mature landfill leachate. Parameter

Value (mean ± S.D.)a

Parameter

Value (mean ± S.D.)a

COD BOD5 NH4+-N TN

1026.8 ± 14.2 16.7 ± 5.4 2004.8 ± 14.7 2024.5 ± 23.6

NO2−-N NO3−-N Alkalinity pH

< 1.0 < 3.0 6576.3 ± 64.9 8.4 ± 0.3

a

2

Values are in mg L−1 except pH.

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(10 min) and discharge (10 min) periods were carried out once a day. The schematic diagram and experimental procedure were detailed in Supplementary Material. The effects of influent loads on nitrogen and COD removal from the raw leachate were investigated in this study. Based on the influent nitrogen and COD loads, the experiment consisted of three stages. At stage I, stage II, and stage III, influent nitrogen loads were 200.5 ± 1.5, 300.5 ± 2.2, and 401.3 ± 3.0 g N m−3 d−1, respectively, and influent COD loads were 102.6 ± 1.5, 154.1 ± 2.1, and 205.5 ± 2.8 g COD m−3 d−1, respectively. Corresponding volume exchange ratios were 0.10, 0.15, and 0.20 at stage I, stage II, and stage III, respectively.

DOMFluor toolbox (Stedmon and Bro, 2008), which could identify outlier samples as well as perform split-half analysis and residual errors diagnostics (Li et al., 2014).

FI = (I370/450)/(I370/500)

Where I370/450 and I370/500 represented fluorescence intensities at Ex/ Em of 370 nm/450 nm and 370 nm/500 nm, respectively.

HIX = ( I 435 - 480/255)/( I300 - 345/255)

2.3.5. In-situ assays of maximum Anammox and denitrification activities Assays of maximum Anammox activity (rAMX,max) and denitrification activity (rDN,max) were performed according to a previous work (Laureni et al., 2016). The rAMX,max was defined as the volumetric consumption rate of influent TN, with both of initial NH4+-N and NO2−-N concentrations of 25 mg L−1. The rDN,max was defined as the volumetric consumption rate of NO3−-N, with initial NO3−-N and COD (sodium acetate as the carbon source) concentrations of 50 and 300 mg L−1, respectively. Assays of rAMX,max and rDN,max were conducted in-situ in SNAD bioreactor at the end of the SBBR cycle for 1–2 h, and calculated by linear regression fitting analysis using nitrogen concentrations of three to six grab liquid samples, which was collected in triplicate every 20 min. Values of rAMX,max and rDN,max were measured at day 0 (inoculum), 60 (stage I), 100 (stage II), and 140 (stage III), respectively. In addition, SNAD reactor was washed with tap water to remove the residual nitrogen and organic matter before and after each activity assay.

2.3.1. Determination of SNAD effectiveness Liquid samples were collected once a day, and subsequently, the concentrations of nitrogen species and COD were determined according to the standard methods (APHA, 2012). Concentrations of BOD5 were determined by a BOD meter (Hach, BOD Track II, USA). In addition, DO and pH were measured by a multi-parameter (Hach, HQ30D, USA). 2.3.2. Percentage of Anammox contribution to TN removal (NH+4 - Ncon,Amx + NO2 - Ncon,Amx - NO3 - Npro,Amx ) TNcon

(1) Where NH4+-Ncon,Amx and NO2−-Ncon,Amx were the consumptions of NH4+-N and NO2−-N during Anammox process, respectively. The sum of NH4+-Ncon,Amx and NO2−-Ncon,Amx was equal to NH4+-N consumption amount minus NO2−-N accumulation amount (NH4+-Ncon-NO2−Nacc) in SNAD bioreactor. NO3−-Npro,Amx represented theoretic NO3−-N production during Anammox process, which was equal to 0.112 (NH4+Ncon-NO2−-Nacc). TNcon represented determined TN removal amount in SNAD bioreactor. The calculation was based on the assumptions that nitrite-oxidizing bacteria (NOB) was completely inhibited in SNAD bioreactor, and NO3−-N was the ultimate electron donor of denitrification.

2.4. Metagenome sequencing and bioinformatic analysis 2.4.1. Sampling, DNA extraction and metagenome sequencing The sludge samples were collected on day 0 (PNA, inoculum) and 140 (SNAD, stage III), and termed as D0 and D140, respectively. The collected sludge samples were immediately cryopreserved at −80 °C prior to further DNA extraction and metagenome sequencing. Genomic DNA extraction was conducted as described in previous work (Wang et al., 2019). The purity and concentration of the extracted DNA samples were quantified using NanoDrop-2000 (Thermo Fisher Scientific, USA) and TBS-380 (Turner Biosystems, USA), respectively, and the quality was verified using gel electrophoresis with 1% agarose. The extracted genomic DNA was submitted to Majorbio Co., Ltd (Shanghai, China), for metagenome sequencing. The paired-end sequencing was conducted on an Illumina HiSeq 4000 platform (Illumina Inc, USA). Metagenomic assembly, contigs binning and open reading frames prediction were performed according to a previous paper (Speth et al., 2016). The analysis of raw data was conducted on I-Sanger Cloud Platform (www.i-sanger.com).

2.3.3. Percentage of denitrification contribution to NO3−-N reduction

Denitrification percentage =

(NO3 - Npro,Amx

NO3 - Npro )

NO3 - Npro,Amx

(4)

Where Σ I435-480/255 and Σ I300-345/255 represented the sum of fluorescence intensities at Ex/Em of 435–480 nm/255 nm and 300–345 nm/ 255 nm, respectively.

2.3. Sampling and analysis methods

Anammox percentage =

(3)

(2)

Where NO3−-Npro represented determined NO3−-N production in SNAD bioreactor. It was assumed that NOB did not contribute NO3−-N production in SNAD bioreactor, and NO3−-N was the ultimate electron donor of denitrification. 2.3.4. Spectroscopy analysis of dissolved organic matters (DOM) The three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectra of DOM in the mature landfill leachate and effluents of SNAD bioreactor during different stages were performed by a luminescence spectrometer (Hitachi, F-7000, Japan). The scanning field of excitation (Ex) wavelength increased from 200 to 450 nm, and emission (Em) wavelength increased from 280 to 550 nm, with 5 nm of intervals for both Ex and Em modes. The scanning speed of the 3D-EEM measurements was set at 1200 nm min−1. Moreover, two fluorescence indices including fluorescence index (FI) and humification index (HIX) were calculated based on previous work (Sun et al., 2014). FI can be used to distinguish the origin of DOM (Eq. (3)). FI values over 1.90 indicates that DOM is microbially derived, while those below 1.40 refers to terrestrially derived DOM. HIX is an index to assess the humification, and a higher HIX means a higher humification degree of DOM (Eq. (4)). In addition, parallel factor analysis (PARAFAC) was conducted by Matlab R2017a (Mathworks, Natick, MA, USA) with the

2.4.2. Pathways of nitrogen and carbon cycling metabolisms To obtain comprehensive bioinformatic pathways concerning nitrogen and carbon cycling metabolisms for mature landfill leachate treatment in SNAD bioreactor, the sequences of the predicted genes in each sludge sample were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/keeg/), and “KEGG Viewer” modules in MEGAN were used to obtain the bioinformatic pathways concerning nitrogen cycling and aromatic organic compounds degradation. For these taxonomic annotations, sequences of the non-redundant gene catalog were aligned to NCBI-NR annotations with the maximum e-value cutoff of 10−5 with BLASTP (Version 2.2.28+). In this study, pathways of nitrogen metabolism (pathway ID: ko00910) and degradation of aromatic compounds (pathway ID: ko01220) were obtained from “KEGG Viewer” modules. For nitrogen metabolism, this work mainly focused on the linkages between microbial community and 3

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Fig. 1. Variations in effluent (a) N species and (b) COD concentrations; removal loads and rates of (c) nitrogen and (d) COD during stage I, II and III; and variations in (e) determined NO3−-Npro in the effluent, theoretic NO3−-Npro by PNA and NO3−-Ncon by DN, and (f) ratios of CODcon/NO3−-Ncon by DN.

functional genes for nitrification (amoA and hao), Anammox (hdh, hzsA, hzsB and hzsC), denitrification (napA, napB, narG, narH, nirK, nirS, nirB, nirD, norB, norC and nosZ) and dissimilatory nitrate reduction to ammonium (DNRA, nrfA, nrfH). Furthermore, functional genes including ubiX, bcrB, dch, catE, dmpC, pcaC, adhP, yiaY, frmA and hcaC, and the annotated microorganisms for aromatic compounds degradation were analyzed, when considering that humic-like substances were the dominant soluble DOM in mature landfill leachate in this work.

Additionally, their metabolic pathways and target chemicals were detailed in Supplementary Material. 3. Results and discussion 3.1. Nitrogen removal and stoichiometric analysis of SNAD system Fig. 1a shows that nitrogen removal efficiency of the SBBR 4

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significantly decreased during the initial 7 days in stage I after fed with mature landfill leachate. TN removal loads decreased from 170.6 ± 1.9 mg L−1 d−1 to 86.1 ± 2.3 mg L−1 d−1. Variations in effluent NH4+-N and NO2−-N concentrations significantly increased, whereas those in the effluent NO3−-N concentration presented the opposite trend (Fig. 1a). After a short-term acclimatizing period, nitrogen removal effectiveness gradually recovered, and subsequently tended to be stable since the 43rd day (Fig. 1a), with NH4+-N and TN removal rates of 99.9 ± 0.1% and 94.9 ± 0.4%, respectively (Fig. 1c). These results demonstrated that mature landfill leachate posed inhibitory effects on the single-stage SBBR. However, the activities of PN and Anammox could be recovered to a great extent after long-term acclimatizing. When influent nitrogen loads increased, variations in nitrogen removal efficiency during stage II and stage III were similar to those during stage I (Fig. 1a). However, the acclimatizing periods during stage II (15 d) and stage III (17 d) were shorter, when compared that during stage I. NH4+-N and TN removal efficiencies during stage II were 99.2 ± 0.2% and 91.4 ± 0.3%, respectively, and those during stage III were 98.9 ± 0.1% and 90.7 ± 0.2% in the single-stage SBBR, respectively (Fig. 1c). According to previous work, TN removal efficiency could not exceed 88.8% in PNA process owing to 11.2% of residual NO3−-N in the effluent (Bagchi et al., 2012; Wang et al., 2018). In this study, the ratios of NO3−-Npro/(NH4+-Ncon + NO2−-Ncon) (where pro and con are the abbreviations for production and consumption, respectively) were 5.2 ± 0.4%, 8.4 ± 0.3% and 8.8 ± 0.2% during stage I, stage II and stage III, respectively (Fig. 1a), which were lower than 11.2%. Such phenomenon showed that the indicated that the actually formed NO3−N was significantly lower than the theoretically formed NO3−-N, suggesting that part of the formed NO3−-N by PNA process might be removed through other pathways. Assay for maximum denitrification activity showed that values of rDN,max significantly increased after longterm acclimating to mature landfill leachate, when compared with those fed with synthetic ammonium-rich wastewater (p < 0.01, Table 2). This demonstrated that (partial) denitrifiers could facilitate NO3−-N removal in the single-stage SBBR, in spite of negligible biodegradable organics in the mature landfill leachate. Refractory organic compounds or their metabolites in mature landfill leachate might serve as the potential electron donor for denitrification in the system. To conclude, nitrogen eliminated from mature landfill leachate in the intermittently aerated SBBR might be ascribed to simultaneous partial nitrification, Anammox and (partial) denitrification (SNAD) process. Stoichiometric calculation indicated that PNA process contributed to 96.1 ± 2.0%, 96.2 ± 0.7% and 97.2 ± 0.8% of TN removal during stage I, II and III, respectively. This demonstrated that PNA dominated TN removal from mature landfill leachate in SNAD bioreactor. In addition, Fig. 1e shows that the percentage of denitrification contribution to NO3−-N removal were 54.3 ± 3.5%, 22.9 ± 2.9% and 16.1 ± 2.6% during stage I, II and III, respectively. The percentage of denitrification contribution to NO3−-N removal decreased with higher influent organics loads. This demonstrated that although the refractory organic matters in mature landfill leachate might serve as the potential electron donor for denitrification, they could also inhibit denitrification

activities in SNAD bioreactor. This result was consistent with variations in maximum denitrification activities with different influent organics loads (Table 2). Besides, Fig. 1f illustrated that the ratios of CODcon/ (NO3−-Ncon by DN) were 5.18 ± 0.42, 8.37 ± 0.30 and 8.83 ± 0.30 during stage I, II and III, respectively. This phenomenon might demonstrate that more carbon substances were required as the energy source to drive denitrification process in SNAD system, when exposed to higher concentrated mature landfill leachate. 3.2. Anammox and denitrification activities in response to leachate exposure Assays for maximum Anammox activity demonstrated that the rAMX,max decreased after long-term exposure to mature landfill leachate (Table 2). The relative rAMX,max of SNAD bioreactor decreased to 94.4 ± 5.7%, 86.6 ± 2.5% and 74.0 ± 5.1% during stage I, stage II and stage III, respectively, when compared with that fed with synthetic ammonium-rich wastewater in the inoculum (Table 2). This indicated that the Anammox activities decreased under exposure to higher concentrated mature landfill leachate in SNAD bioreactor. It should be noted that higher concentrations of organic compounds (especially aromatic compounds) had an inhibitory effect on Anammox activity (Ramos et al., 2015), which hindered nitrogen removal from mature landfill leachate. The lipophilic compounds of the aromatics combining with hydrophobic parts of the phospholipid bilayer of the cytoplasmic membrane of Anammox bacteria might pose toxic effects, including the loss of membrane integrity and increase of ions permeability (Sikkema et al., 1995). Assays for maximum denitrification activity demonstrated that the rDN,max significantly increased after fed with mature landfill leachate (p < 0.01). After long-term exposure to mature landfill leachate, values of relative rDN,max of SNAD bioreactor were 2.36–6.16-fold higher than that fed with synthetic ammonium-rich wastewater (Table 2). This demonstrated that denitrification metabolism was significantly enhanced for simultaneous nitrogen and refractory organic compounds removal from mature landfill leachate. Besides, after long-term exposure to the raw leachate, values of rDN,max were inversely proportional to the effluent COD concentrations (R2 = 0.998). Such result indicated that refractory organic compounds might inhibit denitrification activities in SNAD bioreactor, although they could serve as the potential electron donor for denitrifiers. 3.3. COD removal performance of SNAD system In this study, BOD5/COD ratio of the mature landfill leachate was low (~1.6%), which hindered the biodegradation of organic compounds in SNAD bioreactor. However, the bioreactor achieved COD removal rates of 67.7 ± 2.2%, 50.3 ± 1.3% and 46.2 ± 1.1% during stage I, stage II and stage III, respectively (Fig. 1d). The degradation of organic compounds could relieve the inhibitory effects on Anammox and denitrification by mature landfill leachate, which was of significance to the stability of the Anammox-denitrification synergy. However, a higher concentration of COD (330.3–553.6 mg L−1) in the effluent of SNAD bioreactor was observed (Fig. 1b), which exceeded the

Table 2 Maximum Anammox and denitrification activities in the experiment. Stage

Day

rAMX,maxa

Inoculum I II III

0 59–60 99–100 139–140

22.39 21.14 19.39 16.57

a b

± ± ± ±

0.42 1.27 0.55 1.13

Values are in mg L−1 h−1. Values of relative activities are based on that of the inoculum (day 0).

5

Relative rAMX,maxb

rDN,maxa

Relative rDN,maxb

100.0 ± 1.9% 94.4 ± 5.7% 86.6 ± 2.5% 74.0 ± 5.1%

1.51 ± 0.34 10.81 ± 0.13 6.38 ± 0.25 5.08 ± 0.14

100.0 715.9 422.5 336.4

± ± ± ±

22.5% 8.4% 16.8% 9.4%

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Fig. 2. 3D-EEM spectra of DOM in the (a) raw leachate and effluents of SNAD bioreactor during (b) stage I, (c) stage II and (d) stage III; and variations in (e) Fmax and (f) variation ratios of different components by PARFAC analysis.

COD threshold of Standard for Pollution Control on the Landfill Site of Municipal Solid Waste in China. Therefore, in order to meet the stringent discharge standard, enhancement of refractory organic compounds degradation from mature landfill leachate should be addressed, such as advanced oxidation (Anfruns et al., 2013).

3.4. DOM transformations in SNAD system Fig. 2 shows the 3D-EEM spectra of DOM in the raw leachate and effluents of SNAD bioreactor. The fluorescence fingerprint of the raw leachate indicated three peaks at Ex/Em wavelengths of 365 nm/445 nm 6

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(peak A), 320 nm/445 nm (peak B), and 255 nm/460 nm (peak C), respectively (Fig. 2a). These peaks could be characteristic of humic-like substances (Chen et al., 2003). In addition, the 3D-EEM spectra of the effluents showed that the fluorescence location did not significantly shift, but the fluorescence intensity (especially peak A) significantly decreased, when compared with those in the raw leachate (Fig. 2). Such phenomenon indicated that humic-like substances could be degraded in SNAD system. Besides, the increased FI values in the effluents showed that a greater proportion of DOM derived from microbial activity (Supplementary Material), demonstrating that the microorganisms played crucial roles in DOM transformations in SNAD system. The HIX values in the effluents of SNAD system were lower than that in the raw leachate (Supplementary Material), suggesting that DOM in the effluent had a lower degree of humification than that in the raw leachate. This might be ascribed to the degradation of humic substance in SNAD system. To conclude, these findings demonstrated that SNAD system might favor humic acid-like substances removal. Moreover, the PARAFAC modeling identified four fluorescent components. Component 1 (C1), component 2 (C2), component 3 (C3) and component 4 (C4) had maximum Ex/Em wavelengths of 365 nm/ 450 nm, 335 nm/400 nm, 395 nm/485 nm and 255 (310) nm/455 nm, respectively. The fluorescence fingerprint of C1 and C3 and were characterized as UVA humic-like (A), and C2 and C4 were characterized as UVA marine humic-like (M) and UVC humic-like (C) substances (Coble, 2007; Ishii and Boyer, 2012). Variations in Fmax of fluorescent components of the raw leachate and effluents of SNAD bioreactor by PARAFAC modeling are illustrated in Fig. 2e. Relevant research demonstrated that values of Fmax were proportional to the concentrations of DOM in Anammox-based bioreactor (Li et al., 2018b). Therefore, variation rates of different components during different periods were calculated based on variations in Fmax. The PARAFAC results showed that the concentrations of C1, C2 and C3 in the effluents decreased by 42.5–47.7%, 21.8–25.5% and 54.0–60.4%, respectively, when compared with that in the raw leachate (Fig. 2f). Such phenomenon indicated that SNAD bioreactor favored UVA humic-like and UVA marine humic-like degradation. In contrast, the concentrations of C4 in the effluents of SNAD system were 47.0–55.1% higher than that in the raw leachate, implying that the incomplete degradation of humic-like occurred. It has been demonstrated that the molecular size of DOM was positively associated with the peak wavelengths of Ex and Em (Wu et al., 2003). Thus, fluorophore responsible for C4 was expected to consist of relatively small molecular size DOM. In addition, the physiochemical behavior of C4 has been reported to be biologically unavailable, whereas those of C1, C2 and C3 could be biologically degraded and produced by certain microorganisms (Ishii and Boyer, 2012). These results indicated that C4 might be certain biological metabolites of C1, C2 and/or C3, and was difficult to be biologically degraded. Considering the unavailable biodegradation characteristic of C4, further physical and/or chemical treatment techniques for UVC humic-like degradation were required in order to meet the COD threshold of discharge standard of landfill leachate.

contrast, the relative abundance of AMX including Candidatus Brocadia, Candidatus Jettenia and Candidatus Kuenenia decreased by 47.5–52.6% (Fig. 3). These results demonstrated that long-term exposure to mature landfill leachate of SNAD system would suppress the growth of AMX rather than AOB. The growth of NOB such as Nitrospira was almost completely inhibited by mature landfill leachate (Fig. 3), implying that the raw leachate posed inhibitory effects on NOB growth, and NO3−-N produced in SNAD bioreactor was predominantly derived from Anammox process. In addition, the remaining 17 genera in the SNAD system were affiliated with phyla Chloroflexi (7 genera, 21.32%), Proteobacteria (4 genera, 1.48%), Ignavibacteriae (2 genera, 8.82%), Chlorobi (1 genus, 5.12%), Bacteroidetes (1 genus, 0.81%), Armatimonadetes (1 genus, 0.45%) and Acidobacteria (1 genus, 0.26%) (Fig. 3). The microbial community by the draft genomes indicated that most of these bacteria have been detected in other Anammox-based systems (Supplementary Material), implying that this research has broad relevance for investigating the microbial community in SNAD system for the treatment of mature landfill leachate and similar industry wastewater. However, except AMX, AOB and NOB, the metabolic potential and ecosystem functioning of these organisms has not been extensively investigated for mature landfill leachate treatment in SNAD system (Li et al., 2018a; Liang et al., 2014). Therefore, identification of the linkage between functional genes and microbial ecology in SNAD system for mature landfill leachate treatment is needed, which could potentially enhance simultaneous nitrogen and refractory organics removal from mature landfill leachate by addressing microbial constraints. 3.5.2. Potential of nitrogen conversions in SNAD system As SNAD bioreactor was an ammonium-driven ecosystem, the genomes encoding key enzymes related to nitrogen cycle were annotated in the study. The key enzymes and annotated microorganisms relevant to PN, Anammox and denitrification in the study were illustrated in Fig. 4a–c. Taxonomic annotations indicated that bacteria affiliated with genus Nitrosomonas encoding ammonium monooxygenase (amoA) and hydroxylamine oxidoreductase (hao) contributed to aerobic ammonium oxidation (Fig. 4a). Besides, no ammonium-oxidizing archaea encoding amoA was detected in the system. The sequence numbers of amoA and hao increased from 10,016 and 22,468 of sample D0 to 14,818 and 30,142 of sample D140, respectively. However, the sequence numbers of hydrazine dehydrogenase (hdh) and hydrazine synthase (hzs, including hzsA, hzsB and hzsC) derived from Candidatus Brocadia and Candidatus Kuenenia decreased from 24,772 and 30,482 in sample D0 to 15,126 and 19,180 in sample D140, respectively (Fig. 4b). These results demonstrated that AOB could better acclimatize to the ecosystem after fed with mature landfill leachate, when compared with AMX. Fig. 4c shows that the sequence numbers of key denitrification enzymes (except napA and nirS) increased by 7.8–47.9% after long-term exposure to mature landfill leachate, which was correlated to variations in maximum denitrification activities (Table 2). In SNAD bioreactor, nitrate reductase subunit alpha (narG) was mainly derived from Armatimonadetes bacterium CSP1-3, uncultured Acetothermia bacterium and Acidovorax oryzae. Nitrate reductase subunit beta (narH) was derived from Lysobacter daejeonensis and Elioraea tepidiphila. Nitrite reductase (NO forming, nirK) was derived from Ardenticatena maritima and Rhodanobacter sp. Root627. Nitric oxide reductase subunit B (norB) was derived from Mesorhizobium sp. Root157. Nitric oxide reductase subunit C (norC) was derived from Sediminibacterium sp. C3. Nitrous oxide reductase (nosZ) was derived from Chloroflexi bacterium CSP1-4, Runella limosa and Candidatus Acetothermus autotrophicum. In addition, nitrite reductase (cytochrome c-552, nrfA) and cytochrome c nitrite reductase small subunit (nrfH), as key enzymes of DNRA process, was derived from Ignavibacterium album and Chloroflexi bacterium OLB13, respectively (Fig. 4c). Besides, denitrification enzymes including napB, nirB and nirD were rarely detected (reads number < 1000) in the SNAD bioreactor.

3.5. Microbial community succession and bioinformatic analysis 3.5.1. Microbial community overview To obtain a genome-scale microbial community succession after fed with mature landfill leachate, metagenomic sequencing was performed using representative DNA samples from the PNA and SNAD bioreactors. Taxonomic affiliation based on contigs demonstrated that bacteria were the predominant domain, accounting for more than 99.0% of DNA sequences. In addition, metagenomic sequencing demonstrated that 18 and 21 bacterial genera dominated (> 0.1%) in samples of D0 and D140, respectively (Fig. 3). The predominant bacteria responsible for the key metabolisms of PN and Anammox processes were Nitrosomonas (AOB) and Candidatus Brocadia (AMX), respectively. The relative abundance of Nitrosomonas increased from 15.48% (D0) to 17.92% (D140) (Fig. 3). In 7

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Fig. 3. Abundance of the 22 dominant genera and the corresponding phyla in the inoculum and SNAD bioreactor. The genera with the same legend color are assigned to the same phylum.

Given that a higher abundance of denitrification genes was found in SNAD bioreactor, the accumulated NO3−-N by Anammox process might be mitigated by denitrifiers which catalyze the conversions of NO3−-N to N2. Besides, NO3−-N could be reduced to NO2−-N by partial denitrifiers and/or further to NH4+-N by DNRA bacteria, which could serve as metabolic substrates of Anammox for TN removal (Li et al., 2019). Fig. 4c shows that the reads number of nitrate reductase including narG and narH were much more abundant than those of nitrite reductase (e.g., nirK), nitric oxide reductase (e.g., norB) and nitrous oxide reductase (e.g., nosZ), suggesting that partial denitrification might play crucial roles in NO3−-N reduction to NO2−-N. To conclude, the occurrence of complete denitrifiers, as well as the synergy of partial denitrifiers, DNRA bacteria and AMX, could enhance TN removal efficiency from mature landfill leachate in SNAD system.

communis and Aminiphilus circumscriptus might facilitate humic-like substances degradation from mature landfill leachate in SNAD bioreactor. The phyla Chloroflexi and Chlorobi have been extensively found to interact with Planctomycetes (Lawson et al., 2017; Liu et al., 2017; Pereira et al., 2017), and their distributions in Anammox-based bioreactor were summarized in Supplementary Material. In the Anammox and PNA system, the roles of organisms affiliated with Chloroflexi and Chlorobi in Anammox biofilm were to utilize and catabolize the decaying Anammox bacterial cell materials and extracellular peptides in the EPS matrix under anoxic conditions, with the produced NO3−-N during Anammox process as the electron acceptor (Kindaichi et al., 2012; Lawson et al., 2017). Moreover, organisms affiliated with Chloroflexi and Chlorobi have been reported to be capable of aromatic compound (e.g., humic-like substances) degradation (Colatriano et al., 2018; Stern et al., 2018), which might play key roles in refractory organic matters removal from mature landfill leachate in SNAD system. Therefore, given that the organisms affiliated with Chloroflexi and Chlorobi were often observed in the PNA systems fed with synthetic ammonium-rich wastewater (Supplementary Material), these organisms replete with aromatics degradation genes might facilitate the occurrence of refractory organic compounds removal after mature landfill leachate was added. In addition, the degradation of aromatic compounds in SNAD could alleviate the inhibitory effects of AMX, which favored the stability of the Anammox-based reactor. These results demonstrated that biomass of the synergy of AMX and its co-existent heterotrophs including Chloroflexi and Chlorobi in Anammox-based system could be an applicable inoculum for simultaneous nitrogen and refractory organic compounds removal from mature landfill leachate and similar industry wastewater. The bioinformatic results demonstrated that Nitrosomonas eutropha and Nitrosomonas communis might facilitate aromatic compounds degradation from mature landfill leachate, although these organisms have been widely recognized to possess the AOB phenotype. This indicated that several AOB were capable of using a mixotrophic mode when exposure to aromatic organic compounds in mature landfill leachate, making them potential candidates for bioremediation of refractory organic compounds. This might be the reason that AOB could acclimated well to long-term mature landfill leachate exposure.

3.5.3. Genes expression of aromatic compounds conversions Bioinformatic analysis of aromatic compounds biodegradation in mature landfill leachate is of significance to investigate DOM conversion pathways and its interactions with nitrogen removal in SNAD system, as humic-like substances were the dominant degraded organic compounds in SNAD bioreactor for mature landfill leachate treatment. In total, 90 and 95 genes implicated in aromatic compounds conversions were detected in samples D0 and D140, respectively. In this study, the metabolic pathways of aromatic compounds conversions mainly included dioxygenase and dehydrogenase reactions, meta-cleavage of catechol, ring cleavage via beta oxidation, and conversion on aromatic ring (Fig. 4d). The sequence numbers of the key enzymes relevant to aromatic compounds conversions increased by 28.3–157.1% after long-term exposure to the raw leachate, demonstrating that the enrichment of functional bacteria responsible for aromatic compounds biodegradation was achieved in SNAD bioreactor. The metabolism of dioxygenase and dehydrogenase reactions was implicated by the detection of adhP, yiaY, frmA and hcaC enzymes, which were mainly derived from Chloroflexi bacterium OLB14, Nitrosomonas eutropha and Aminiphilus circumscriptus in SNAD bioreactor (Fig. 4d). The key enzymes relevant to meta-cleavage of catechol were catE, dmpC and pcaC, which were mainly derived from Chloroflexi bacterium OLB14, Ignavibacterium album and Chlorobi bacterium OLB6. Moreover, the main anaerobic metabolisms of aromatic substances in SNAD system included conversion on aromatic ring and ring cleavage via beta oxidation, which were implicated by the detection of dch, bcrB and ubiX enzymes (Fig. 4d). Taxonomic annotations indicated that organisms affiliated with phylum Chlorobi and species Nitrosomonas communis facilitated anaerobic degradation of aromatic compounds. Overall, the metagenome-based microbial ecology illustrated that organisms affiliated with phyla Chloroflexi and Chlorobi, as well as species Ignavibacterium album, Nitrosomonas eutropha, Nitrosomonas

3.5.4. Inferred ecological model for simultaneous nitrogen and carbon removals In this study, SNAD bioreactor achieved simultaneous nitrogen and refractory organic compounds removals from mature landfill leachate, and the inferred ecological model was illustrated in Fig. 5. The synergy of genera Nitrosomonas and Candidatus Brocadia were the key organisms 8

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Fig. 4. Profiles of (a) AOB-related, (b) Anammox-related, (c) denitrification-related, and (d) aromatic compounds conversions-related genes and their annotated microorganisms in the inoculum and SNAD bioreactor.

responsible for TN removal owing to the metabolisms of PN and Anammox processes, respectively. Besides, the NO3−-N accumulated intrinsically during Anammox process could be reduced by denitrifiers. On one hand, NO3−-N could be completely reduced to N2 owing to the catalysis by the key denitrification enzymes including narG/narH, nirK/ nirS, norB/norC and nosZ. On the other hand, the accumulated NO3−-N might be mitigated by partial denitrifiers implicated by narG/narH enzyme, and/or further utilized by DNRA bacteria implicated by nrfA/ nrfH enzyme (Fig. 5). The produced NO2−-N and NH4+-N during partial denitrification and DNRA processes could serve as metabolic substrates for Anammox, and the “nitrite loop” and/or “ammonium loop” could enhance TN removal rate in SNAD system. In addition, the electron donor for denitrification and DNRA might be derived from humic-like substances in the mature landfill leachate as well as their intermediates. The metabolic pathways of these organic compounds conversions mainly included dioxygenase and dehydrogenase reactions (adhP, yiaY, frmA and hcaC enzymes), meta-cleavage of catechol (catE, dmpC and pcaC enzymes), ring cleavage via beta oxidation (dch, bcrB

enzymes), and conversion on aromatic ring (ubiX enzyme), which might be ascribed to the organisms affiliated with phyla Chloroflexi and Chlorobi, as well as genera Ignavibacterium, Nitrosomonas and Aminiphilus (Fig. 5). Considering that these organisms were relatively abundant in the PNA bioreactor fed with synthetic ammonium-rich wastewater, the synergy of AOB, AMX and their co-existing heterotrophs in Anammox-based bioreactor could be an applicable inoculum for the construction of SNAD bioreactor for the treatment of mature landfill leachate and similar industry wastewater. The inferred ecological model provided bioinformatic roles for the previously understudied microorganisms in SNAD bioreactor for mature landfill leachate treatment. However, it should be mentioned that the model was based on the variations in the sequence numbers of key genes which facilitated nitrogen and aromatic compounds conversions. Therefore, further validation such as metaproteomic or isolation of key functional microorganisms from this bioreactor is needed, which is essential to enhance nitrogen and refractory compounds removals from mature landfill in SNAD bioreactor by addressing microbial constraints. 9

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Fig. 5. Inferred schematic overview of simultaneous (a) nitrogen and (b) aromatic compounds conversions in SNAD for mature landfill leachate.

4. Conclusions

References

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Acknowledgements This research has been carried out with the financially support from Major Science and Technology Program for Water Pollution Control and Treatment (Grant No. 2012ZX07307-002); Chongqing Science and Technology Commission (Grant No. cstc2018jscx-mszdX0070); and Graduate Scientific Research & Innovation Foundation of Chongqing (Grant No. CYB17005). The author would like to appreciate the contributions of co-workers over the years. Declarations 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. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122166. 10

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