Robustness and microbial consortia succession of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process for mature landfill leachate treatment under low temperature

Accepted Manuscript Title: Robustness and microbial consortia succession of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process for mature landfill leachate treatment under low temperature Authors: Yingmu Wang, Benzhou Gong, Ziyuan Lin, Jiale Wang, Jianbing Zhang, Jian Zhou PII: DOI: Reference:

S1369-703X(18)30010-X https://doi.org/10.1016/j.bej.2018.01.009 BEJ 6858

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

24-8-2017 5-12-2017 7-1-2018

Please cite this article as: Yingmu Wang, Benzhou Gong, Ziyuan Lin, Jiale Wang, Jianbing Zhang, Jian Zhou, Robustness and microbial consortia succession of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process for mature landfill leachate treatment under low temperature, Biochemical Engineering Journal https://doi.org/10.1016/j.bej.2018.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Robustness and microbial consortia succession of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process for mature landfill leachate treatment un-

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der low temperature

Yingmu Wang a, Benzhou Gong a, Ziyuan Lin a, Jiale Wang a, Jianbing Zhang a,

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Jian Zhou a, *

Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Minis-

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try of Education, Chongqing University, Chongqing 400045, PR China

Corresponding author at: Key Laboratory of the Three Gorges Reservoir Region’s

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*

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Eco-Environment, Ministry of Education, Chongqing University, Chongqing Uni-

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versity, Chongqing 400045, China. Tel./fax: +86 23 65120980.

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E-mail address: [email protected] (J. Zhou)

Graphical Abstract

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Highlights 

Robust denitrogenation capability was observed even at 10℃ by PN/ANAMMOX

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SNAD was feasible for efficient TN removal of mature leachate at low temperature Denitrification enhanced TN elimination for mature leachate treatment

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Remarkable bio-refractory organics degradation was conductive to system stabil-

Microbial consortia succession was discussed in detail

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ity

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Abstract:

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The purpose of this research was to assess the feasibility of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) for mature landfill leachate

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treatment under low temperature. To this end, bench-scale bioreactors receiving

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ammonium-rich wastewater were adapted to progressively reduced temperature after 370-day operation (Period I), and subsequently acclimatized to raw leachate for 86

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days (Period II). Maximum nitrogen removal rates (NRR) of 218.9, 211.9, 201.1 and 146.9 g N·m-3·d-1 were achieved at 30°C, 20°C, 15°C and 10°C respectively during

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Period I, mainly ascribing to partial nitritation/ANAMMOX. Furthermore, the SNAD bioreactor turned out to be feasible for prolonged treatment of mature landfill leachate under 15 ℃ (Period II), with simultaneous degradation of nitrogen (207.9 g N·m-3·d-1) and bio-refractory organics (94.9 g COD·m-3·d-1) respectively.

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High-throughput sequencing implied that better denitrogenation performance during

Period II might be ascribed to reappearance of denitrifying bacteria (9.88%) and complete suppression of nitrite oxidation bacteria (NOB), although ammonium oxidation bacteria (AOB) and ANAMMOX bacteria (AMX) decreased by 6.05% and

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4.56% respectively. Furthermore, the enriched bio-refractory organics degradation bacteria (24.38%) were conductive to the performance robustness. This research

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presents perspective for the application of efficient treatment of mature landfill leachate under low temperature.

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Keywords: Mature landfill leachate; Low temperature; Single-stage reactor; SNAD;

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Bio-refractory organic elimination; High-throughput sequencing

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1. Introduction The sanitary landfill remains the primary approach for the ultimate disposal of municipal solid wastes (MSW) on account of its economic advantages [1, 2]. However, leachate generated in municipal landfill sites may contain highly concentrated or-

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ganics and ammonium (NH+4 -N), thus represents a potential source of contamination (e.g., eutrophication) [3]. Characteristics of leachate depend on various factors, e.g.,

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the composition of municipal refuse [4], the age of landfill [5] and climate condition

of landfill region [6]. Particularly, the composition varies largely hinging on the op-

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eration age [7], and leachate generated in mature landfill represented notably poor

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biodegradability, which significantly impeded the biological elimination of nitrogen

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and organics [8].

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Several investigations have demonstrated the possibility of landfill leachate

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treatment by conventional nitrification/denitrification processes [9]. However, the lack of sufficient readily biodegradable organics as electron donors in mature leach-

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ate makes it difficult to satisfy the denitrification process [10]. In addition, it’s une-

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conomic and environment-hazardous due to the external organic carbon dosing. Recently, the integration of anaerobic ammonium oxidation (ANAMMOX) and partial nitrification (PN) have proved to meet the significant demands for nitrogen elimina-

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tion of mature landfill leachate [11]. The coupling of PN/ANAMMOX, compared with nitrification/denitrification, allows 63% lower aeration supply, no need of external carbon source dosing and lower sludge yield [12]. However, the application of PN/ANAMMOX for mature leachate treatment was 4

impeded by the bottleneck of the instability under low temperature and a limited view of microbial ecology in the processes. It is generally recognized that the growth of ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and ANAMMOX bacteria (AMX) vary notably with temperature fluctuation. Relative

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studies have demonstrated that the optimum temperature range for the processes was approximately 30-40 ℃ [13]. Within the temperature range, AOB represented a sig-

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nificantly larger growth rate than NOB. A low temperature tends to narrow the

growth gap between AOB and NOB, which inhibits partial nitrification process. In

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addition, AMX could represent biologically efficient capability and better stability

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under a moderately thermophilic condition (>30 ℃). Interestingly, recent investiga-

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tions with marine samples reported measurable ANAMMOX activity at low temper-

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ature (<20 ℃). Rysgaard, Glud, Risgaard-Petersen and Dalsgaard [14] demonstrated

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the maximum activity of ANAMMOX in sediments on the coast of Greenland at 12 ℃. Similar observation was found by Dalsgaard [15] in sediments from Bal-

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tic-North Sea near arctic. Moreover, several lab-scale studies reported the ANAM-

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MOX activity below 20 ℃ for wastewater treatment [16]. Moreover, the environmental stress (e.g., concentrated harmful organics, heavy metals and xenobiotics) might have negative on microbes responsible for nitrogen elimination [17]. Consid-

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ering that little effort for mature leachate treatment has been reported below 20 ℃ and the complexity of raw leachate, it was worthy of investigating the denitrogenation performance and microbial ecology for mature landfill leachate treatment under low temperature (<20 ℃) based PN/ANAMMOX processes. 5

Theoretically, only 89% of nitrogen removal in maximum could be achieved by PN/ANAMMOX as about 11% of nitrate (NO¯3 -N) remaining in the effluent. To satisfy the stringent total nitrogen (TN) discharged standard, any effort to enhance nitrogen contaminant elimination should be addressed. Alternatively, the integration of

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PN/ANAMMOX and heterotrophic denitrification in a single-stage reactor, also recognized as SNAD process, could meet the demands for residual nitrate and or-

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ganics removal [18] . However, considering the notably poor biodegradability of

mature leachate in this work, whether the bio-refractory organics could serve as po-

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tential electron donors for denitrification would be investigated.

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Drawing on the previous researches, sequencing batch biofilm reactors (SBBRs)

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receiving synthetic ammonium-rich wastewater were operated under stepwise de-

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creasing temperatures from 30 to 10 ℃ for 370 days (Period I), and subsequently fed

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with mature landfill leachate under low temperature (15 ℃) for 86 days (Period II). The main aims of this work were to: (1) evaluate the feasibility of PN/ANAMMOX

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for autotrophic nitrogen removal of mature landfill leachate at low temperature; (2)

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investigate whether the bio-refractory organics in leachate could serve as potential electron donors for heterotrophic denitrification. To accomplish these, performance of nitrogen (i.e., ammonium, nitrite, nitrate) and chemical oxygen demand (COD)

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elimination were assessed by mass balance. Moreover, the succession of microbial consortia in the systems was characterized by 16S rDNA fragment sequencing.

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2. Materials and methods 2.1. Synthetic wastewater and raw leachate In this study, the hypothermic systems were established with temperature declining progressively fed with synthetic ammonium-rich wastewater. The components of

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synthetic wastewater mainly contained NH+4 -N and trace element solution. The NH+4 -N concentration was controlled at approximately 2000 mg·L-1 using NH4HCO3. The

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medium solution contained (in L-1): 25 mg KH2PO4, 6.25 mg FeSO4, 0.3 g CaCl2, 0.2 g MgSO4·7H2O, 0.18 g KHCO3, 1.25 mL trace element solution. The composi-

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tion of the trace element solution was (in L-1): 0.99 g MnCl2·4H2O, 0.43 g

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ZnSO4·7H2O, 0.25 g CuSO4·5H2O, 0.24 g CoCl2·6H2O, 0.22 g Na2MoO4·2H2O,

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0.19 g NiCl2·6H2O, 0.05 g Na2WO4·2H2O, 0.014 g H3BO4.

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The mature leachate utilized in this experiment was collected from the Chang-

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shengqiao landfill (N: 29° 30′ 40.99″, E: 106° 36′ 56.84″) sited in Chongqing which has been put into operation since 2003. The raw leachate had a notably low

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BOD/COD ratio of 0.024 in average, featuring poor in biodegradation. The charac-

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teristics of the raw leachate were detailed on Table 1.

2.2. Reactor and experimental procedure

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In this study, two SBBRs (Fig. 1) were constructed of Plexiglas, and each of them had an effective capacity of 10.0 L, with height of 500 mm and internal diameter of 200 mm. Semi-soft fibrous products were utilized as biomass carriers, and the packing fraction was controlled at 50% (V/V). The experiment was conducted in a ther-

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mostatic chamber for temperature control. Intermittent aeration was carried out in an 8-h cycle sequentially, with aeration stage and non-aeration stage operating alternately for about 4 h and 4 h, respectively (Fig. 1). Dissolved oxygen (DO) was controlled to 2.0-2.4 mg·L-1 at aeration stage by adjusting the aeration intensity of

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air-compressors. Discharging (10 min) and feeding (10 min) were conducted once a day. The volume exchange ratio was 0.125 and influent load was 250 g N·m-3·d-1

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unless specified.

The experiment consisted of two periods (Period I: construction of hypothermic

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systems; Period II: acclimatizing to mature landfill leachate under 15 ℃). In Period I,

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each SBBR was fed with synthetic ammonium-rich wastewater as detailed in Section

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2.1. Based on the reactors (two reactors in a parallel operation condition) in the pre-

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vious published literature [19], PN/ANAMMOX processes were constructed as a

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start-up period with stepwise declining temperature from 30 to 15 ℃. Afterward, the temperature of reactor 1 was further reduced to 10 ℃ to investigate the effect of

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temperature on PN/ANAMMOX, while reactor 2 was fed with raw leachate to in-

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vestigate the feasibility of PN/ANAMMOX on mature leachate under 15 ℃.

2.3. Sampling and chemical analysis

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Water samples were taken and measured twice a day during Period I, while once a day in Period II. Parameters of nitrogen (i.e., NH+4 -N, NO¯2 -N, NO¯3 -N, TKN and TN) and COD was analyzed by the standard methods [20]. BOD5, pH and DO were measured by BOD meter (Hach BODTrak II, USA), pH meter (Hach HQ30D, USA)

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and DO meter (Hach HQ30d, USA), respectively.

2.4. Microbial examination Sludge samples 1#-5# were collected from the SBBR systems on day 12 (30 ℃), day 124 (20 ℃), day 260 (15 ℃), day 370 (10 ℃) during period I and on day 86 (15 ℃)

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during Period II respectively. Samples were cryopreservation under -40℃ until re-

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quired for further molecular biological tests.

Three freeze-thawing cycles with hyperhaline extraction buffer followed by lysis process at 65 ℃ were carried out to extract DNA of sludge samples [21]. The lysis

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process was accelerated by using the intermixture of sodium dodecyl sulfate (SDS),

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cetyltrimethylammonium bromide (CTAB) and proteinase K.

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Before pyrosequencing, PCR amplification of each sludge sample was carried out with a cluster of broad-range gene primers (338F 5′-ACT CCT ACG GGA GGC

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AGC AG-3′, 806R 5′-GGA CTA CHV GGG TWT CTA AT-3′) amplifying the V3-V4

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region. DNA amplification was subsequently performed as the following program: 94 ℃ for 5 min (initial denaturation); 30 cycles of 94 ℃ for 45 s, 60 ℃ for 45 s and

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72 ℃ for 90 s (denaturation, refolding and extension respectively); 72 for 10 min (final extension). Amplification was verified by electrophoresis of the fragments in

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2.0% (wt/v) agarose gels with QuantiFluorTM-ST system (Promega, USA). After the identification of amplification, PCR products were sequencing based on Illumina plateform by Majorbio (Shanghai, China). Similarity retrieval of sequencing could be carried out by comparing with SILVA databases (https://www.arb-silva.de/).

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3. Results and discussion 3.1. Period I: construction of hypothermic systems 3.1.1. Nitrogen removal analysis

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To acclimatize to hypothermic condition, the temperature of PN/ANAMMOX cultures were lowered stepwise from 30 to 10 ℃. The PN/ANAMMOX reactors could

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display stable nitrogen elimination capacity as temperature dropped progressively.

Fig. 2 shows the performance nitrogen turnover during Period I. When the tem-

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perature stepwise reduced from 30 to 20 ℃, no marked drop of nitrogen removal ef-

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ficiency was observed, with NH+4 -N and TN removal ratio slightly decreasing to

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95.4-96.9% and 83.5-84.2%, indicating that the microbes responsible for denitro-

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genation were capable of acclimatizing to the gradually declined temperature condi-

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tion above 20 ℃.

The temperature dropped to 17.5 ℃ and further to 15 ℃ resulted in a drastic drop

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of denitrogenation performance at the initial few days, and the residual NH+4 -N on

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day 186 (164.9 mg·L-1) reached nearly 2.6 folds compared with that under 20℃ (Fig. 2a). This could be explained by the short-term inhibitory effect on AOB and AMX as temperature reduced. Moreover, the outlet NO¯2 -N accumulated slightly (12.4 mg·L-1

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in average, Fig. 2a). Most studies demonstrated that NO¯2 -N accumulation was observed under low temperature [22], and the activity of AMX tended to be more susceptible to lower temperature than that of AOB [23]. Subsequently prolonged operation under 17.5 ℃ and 15 ℃ resulted in recovery and stabilization of denitrogenation 10

activity, with TN removal efficiency of 81.0-82.0% and 79.2-80.2%, respectively. The operational temperature lowered to 12.5 ℃ at day 261 and further to 10 ℃ at day 304 would result in an extreme destabilization of the SBBR (Fig 2). Considering that low temperature could suppress the ammoxidation, the influent loads were re-

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duced for better effluent water quality. As shown in Fig. 2b, a decrease of influent loads seemed to be a feasible approach for the treatment of ammonium-rich

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wastewater at 10℃, since nearly 80% TN elimination was accomplished steadily.

3.1.2. Stoichiometric estimation of AOB, AMX and NOB in autotrophic systems

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The heterotrophic denitrification process could be neglected during period I (based

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on 16S rDNA sequencing, see Sections 3.3), since denitrifying bacteria might have

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been washed out after long-term cultivation without organic carbon source supply in

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the previous experiment. The nitrogen compounds turnover was primarily attributed

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to the microbial consortia of AOB, AMX and NOB. In autotrophic denitrogenation system, the ratio of (NH+4 -Ncon - NO¯2 -Nacc)/ NO¯3

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-Npro (where con, acc and pro are the abbreviations for consumption, accumulation

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and production, respectively) could reveal the proportion of NO¯2 -N consumed by AMX or NOB [24]. The ratio increased with higher activity of consortia of AOB and AMX compared with that of NOB. Note that complete nitrogen removal by the cou-

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pling of AOB and AMX with total suppression of NOB activity would result in the (NH+4 -Ncon - NO¯2 -Nacc)/ NO¯3 -Npro ratio of about 8.90 based on stoichiometric calculation [25]. Fig. 3 shows the revolution of (NH+4 -Ncon - NO¯2 -Nacc)/ NO¯3 -Npro as operation temperature decreased stepwise. Under 30-27.5 ℃, the ratio maintained in nu11

merical value of 8.94±0.07, which was quite approach to value of 8.90. This phenomenon demonstrated that the activity of NOB was suppressed effectively, and the consortia of AOB and AMX outcompeted NOB under mesophilic condition. The ratio tended to drop as the temperature further decreased to 15 ℃. It was

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probably due to the growth gap between AOB and NOB was narrowed, and the activity of AMX was partly suppressed under lower temperature [13, 26]. Relevant lit-

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eratures have reported that the growth rates gap between AOB and NOB varied no-

tably with temperature. The maximum growth rate of AOB decreased from 1.801 d-1

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(30 ℃) to 0.801 d-1 (20 ℃) and further to 0.523 d-1 (15 ℃), while that of NOB repre-

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sented to be 1.182 d-1 (30 ℃), 0.788 d-1 (20 ℃) and 0.642 d-1 (15 ℃), respectively

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[27]. A relatively lower temperature could stimulate a higher proportion of NOB.

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Moreover, AMX was recognized as thermophilic microbes. Thus, deteriorated TN

temperature.

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elimination probably due to the enhancement of nitratation at progressively reduced

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The contribution of nitrogen species turnover by AOB, AMX and NOB was es-

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timated based on a nitrogen mass balance, according to stoichiometric matrix detailed in Supplementary Material (Table S1). The results implied that ANAMMOX process contributed for approximately 82% and 70% nitrite (produced by AOB)

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elimination during operation at 20 ℃ and 15 ℃, respectively. Thus, the integration of AOB and AMX turned out to be the predominant microbes for nitrogen conversion within the temperature range.

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3.2. Period II: acclimatizing to mature landfill leachate under 15 ℃ After acclimatizing to low temperature (15 ℃), the PN/ANAMMOX system was subsequently fed with raw mature landfill leachate for 86 days. The impact of poor biodegradability (BOD/COD=0.024 in average) of leachate on nitrogen elimination

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efficiency was assessed. 3.2.1. COD removal analysis

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During the first 13 days, the COD of effluent raised from 179.5 to 777.2 mg·L-1 (Fig. 4a). The dilution effect, to a great extent, accounted for the illusion COD removal

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due to a relatively low volume exchange ratio of 0.125. It was hypothesized that a

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progressive increase of leachate proportion in the reactor made it possible for mi-

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crobes to acclimatize to such an environment in a relatively short term. Hereafter,

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unexpected COD elimination was observed. The concentration of COD reduced

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gradually to 331.6 mg·L-1 on day 45, and subsequently tended to flatten (331.0±7.3 mg·L-1). The maximum valve of COD removal loading was 94.9 g COD·m-3·d-1 on

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day 64. The valve of BOD5 in outlet was lower than 5 mg·L-1. However, the COD

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consumption far exceeded the BOD5 consumption, and it could imply that about half of bio-refractory organics could be degraded. It was hypothesized that there might existed functional microbes related to elimination of bio-refractory organics in

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leachate, which is conductive to the performance robustness of the SNAD system. The results of 16S rDNA sequencing revealed that microbes capable of bio-refractory organics degradation (e.g., azo dye, aromatic hydrocarbon and phenol), and the succession of microbial consortia was detailed in Section 3.3. 13

3.2.2. Nitrogen removal analysis Besides the persistent COD removal efficiency during period II, an efficient and stable conversion of nitrogen in the SBBR was also observed after nearly 45-day acclimatizing period (Fig. 4b-d). Moreover, the ammonium (NH+4 -N) and total nitrogen

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(TN) turnover performance was comparable to that of system fed with synthetic wastewater at 15 ℃. This phenomenon demonstrated that the microbes responsible

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for denitrogenation process could progressively adapted to the environmental stress

(e.g., high-strength bio-refractory organics, heavy metals and xenobiotics) in raw

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leachate, thus the system could satisfy the demands for efficient nitrogen removal of

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mature landfill leachate under low temperature (15 ℃).

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During the initial 9 d in period II, there existed a relatively great decrease of ni-

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trogen removal efficiency compared with the system fed with ammonium-rich

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wastewater at 15 ℃ (Fig. 4b). The evolution of effluent ammonium and nitrite tended to increase rapidly, while the nitrate concentration in effluent was of the opposite

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trend (Fig. 4c, d). This phenomenon indicated that AOB was inhibited as the propor-

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tion of raw leachate increased, and AMX or NOB was unable to fulfil the complete nitrite conversion. After short-term adaption to raw leachate, the denitrogenation performance tended to recover on days 10-46, with TN removal ratio raised from

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74.2% to 82.4%. Hereafter, the nitrogen compounds in effluent began to flatten, with TN removal ratio fluctuating in the range of 82.1%-82.9%. The average concentration of NH+4 -N in the effluent was 165.4 mg·L-1, while those of NO¯2 -N and NO¯3 -N were 20.8 and 164.6 mg·L-1, respectively. 14

Compared with period I under 15 ℃ (Table 2), the slight decrease of ammonium conversion was detected while nitrite accumulated, indicating the coupling of PN/ANAMMOX processes was inhibited by mature landfill leachate. However, the nitrogen removal efficiency in raw leachate increased by 2.8%, while nitrate com-

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pounds in the effluent decreased by about 40%, as compared to those in period I under 15 ℃.

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In-situ test of maximum heterotrophic denitrification activity on day 80 during

period II was 0.047 g NO-3 -N·L-1·d-1 while negligible denitrification activity was de-

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tected during period I, this demonstrated that heterotrophic denitrification might also

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occurred in the system. The assumption was also supported by the results of 16S

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rDNA sequencing. After acclimatizing to mature landfill leachate under 15 ℃, mas-

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sive heterotrophic denitrifying bacteria (e.g., Denitratisoma, Thermomonas) were

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observed again in the system, although they were eliminated due to 260-day in period I with no carbon source supply (see Section 3.3). The reappearance of denitrify-

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ing microbes in the SBBR probably due to the denitrification activity in raw leachate.

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Moreover, the degraded portion of bio-refractory organics could serve as potential electron donors for denitrification. And relative literature demonstrated that several nitrate-reducing microbes were capable of utilizing specific aromatic compounds as

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sole carbon and energy source for heterotrophic denitrification under oxygen-limited conditions [28]. In addition, the hydrolysis of macromolecular substance by some bio-refractory organisms degradation bacteria might also stimulate the denitrification process [29]. Thus, simultaneous partial nitrification, ANAMMOX and denitrifica15

tion (SNAD) process could be constructed in mature landfill leachate treatment system under low temperature (15 ℃).

3.3. Insight into microbial consortia succession 3.3.1. Bacteria diversity and richness analysis

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In total, 37306, 33399, 30645, 24388, 36462 effective reads were obtained for sludge

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samples 1#-5# respectively. Clustering of Operational Taxonomic Units (OTUs) was carried out on 97% similarity. OTUs numbers among sludge samples 1#-5# repre-

sented to be 144, 157, 161, 127 and 278 respectively. The microbial diversity and

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richness indices were shown in Table 3.

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The rarefaction curves of 5 samples tended to flatten, indicating that the se-

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quencing was enough to cover most of the OTUs (See Fig. S1). The coverage index of each sample exceeded 0.999 on 97% similarity. The Shannon diversity index of

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each sample was below 3.00 except sample 5# of 4.24, and Chao richness index

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showed the similar trend. The microbial consortia of sample 5# exhibited markedly higher diversity and richness characteristics, indicating that organics in the raw could

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probably support more diverse and abundant microbes. It was probably due to a mass of bio-refractory organics degradation in the system and could stimulate the

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rapid growth of heterotrophic bacteria. The temperature exhibited less impact microbial diversity and richness unless under 10 ℃. 3.3.2. PCA analysis The principal component analyses (PCA) of the samples implied that sample 5# exhib-

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ited less similarity with the others (See Fig. 5). It was probably due to distinct culture medium as complicated substrate (e.g., bio-refractory organics) in raw leachate compared with synthetic ammonium-rich wastewater. The principal component 2 (PC2) might represent the impact of temperature on microbial consortia as PC2 score was of

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positive relevance with ambient temperature. PCA analysis implied that the components wastewater was the first principal component affecting bacteria community,

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followed by operational temperature. The PC1 and PC2 accounted for 89.18% of the microbial consortia information.

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3.3.3. Succession of microbes during Period I

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The evolution of microbial community structure was also analyzed at genus taxo-

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nomic units as shown in Fig. 6. The AOB, NOB and AMX in Period Ⅰ were as-

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signed to Nitrosnmonas, Nitrospira and Candidatus Brocadia in genus level, respec-

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tively. Under 30 ℃，the abunbance of AOB and AMX represented to be 16.51% and 9.04%, respectively. Meanwhile, little NOB was detected. The consortia of Nitro-

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somonas and Candidatus Brocadia fulfilled denitrogenation by PN/ANAMMOX

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processes for ammonium-rich wastewater treatment at 30 ℃, corresponding with the results of stoichiometric estimation (see Section 2.1). As temperature decreased, the abundance of AOB, AMX and NOB varied (Fig. 7). When the temperature was

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stepwise lowered from 30 ℃ to 20 ℃ over 112-day cultivation, the relative abundance of AMX decreased to 6.49%, and subsequently reached to 5.96% at 15 ℃, indicating that AMX seemed to acclimatize to cold conditions above 15 ℃. It was probably due to the protection for the activity of AMX in the interior region biofilm 17

with thickness of approximately 9000μm, as shown in Fig. S2. In addition, the AOB made up a predominant fraction of the nitrogen conversion bacteria from 30 ℃ to 15 ℃. The lowered temperature didn’t have significantly adverse effect on ammonium removal, indicating that AOB had a relatively sufficient overcapacity. The rela-

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tively stable coculture of AOB and AMX could contribute to persistent ammonium conversion and efficient nitrogen performance within the temperature range, which

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was similar to a recent research in a nitritation-ANAMMOX bioreactor with temperature dropped from 25 ℃ to 15 ℃ [22]. The abundance of genera Nitrospira was sig-

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nificant higher at 15 ℃ (2.73%) than at 30 ℃ (0.01%) and 20 ℃ (0.53%), resulting in

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increasing proportion of nitrite elimination by NOB as temperature reduced. The

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microbial consortia information demonstrated that the coupling of AOB/AMX out-

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competed NOB above 15 ℃. The temperature further decreased to 10 ℃ resulted in a

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marked decrease of AMX abundance, and the growth gap between AOB and NOB closed. The microbial consortia could fulfil less ammonium conversion and nitrogen

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removal. Considering the deficiency of denitrogenation capability, a mildly reduced

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influent nitrogen loads turned out to be feasible for improving the quality of outlet water at 10 ℃. Efficient nitrogen removal capability probably ascribed to the prolonged acclimation period (370 days) adapting to progressively reduced operational

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temperature even at 10 ℃. 3.3.4. Succession of microbes during Period II Fig. 6a shows the relative abundance of predominant microbial groups at diverse phylum level of 5 various sludge samples. Compared with sample 3#, higher abun18

dance of phyla Chloroflexi, Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria, Armatimonadetes were existed in sample 5#. From the phylum assignment, a relatively significant difference among the sludge samples suggested that the microbial community consortia varied as the characteristics of the fed wastewater

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changed. Except in phylum level, the succession of microbial consortia responsible for

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COD and nitrogen elimination was also analyzed at genus taxonomic units as shown in Fig. 6b and Fig. 7. After 86-day adaption to raw leachate, the genera Comamonas

U

(3.80%), Bellilinea (4.85%), Limnobacter (2.82%), Truepera (1.74%), Brevundimo-

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nas (1.68%), Chloroflexi_uncultured (1.31%), Ferruginibacter (3.04%) and Ther-

A

momonas (6.45%) tended to increase compared with those during period I under

M

15 ℃. The microbes assigned to the above-mentioned genera were reported to be ca-

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pable of bio-refractory organics degradation. The COD removal loads of 73.8 g COD·m-3·d-1 probably ascribed the rise of the above-mentioned microbes. For ex-

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ample, relevant literature demonstrated that some microbes assigned to genera

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Thermomomonas, Comamonas, Bellilinea, Limnobacter and Brevundimonas were capable of degrading bio-refractory organics (e.g., polycaprolactone, azo dye, polychlorinated biphenyls, petroleum) [28, 30-33]. The genus Ferruginibacter could hy-

A

drolysis macromolecular organics, further supplying biodegradable carbon and energy source for other microbes, e.g., heterotrophic denitrifying bacteria [34]. The genus Chloroflexi_uncultured was reported to serve as scavenger for microbial metabolite, which was of significance to the stabilization of treatment process [35]. 19

Truepera could tolerate the environmental resided and hazards in industrial wastewater [36]. The bacteria might be capable of bio-refractory organics degradation jumped about 6 folds after prolonged acclimatization period to mature landfill leachate, resulting in the stabilization of SNAD process treating mature landfill

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leachate. The raw leachate might be a potential source of bio-refractory degradation organisms in the research, as several kinks of microbes capable of eliminating

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bio-refractory compounds in this work were found in raw leachate, e. g., genus

Truepera, family norank_Limnochordaceae of 25.78% and 3.34%, respectively (See

U

Fig. S3).

N

The relative abundance of genera Nitrosomonas and Candidatus_Brocadia

A

dropped by 59.3% and 23.4% respectively, compared with that during Period I under

M

15 ℃. This implied that the AOB and AMX were inhibited by raw leachate to a cer-

ED

tain extent. However, the genera Nitrospira was completely suppressed as the relative abundance of that reduced by 99.9%. It might be ascribed to the multiplying

PT

concentration of free ammonia (FA). During Period Ⅱ, the calculated FA concentra-

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tion increased to 8.18±0.86 mg·L-1 in the effluent [37], surpassing the threshold which NOB would be inhibited. The calculated free nitrous acid (FNA) in Period Ⅱ didn’t inhibit NOB growth. Moreover, the complex compounds in leachate were po-

A

tential environmental stress for NOB. Furthermore, thanks to the reappearance of typical heterotrophic denitrifying bacteria (e.g., Denitratisoma), better TN removal performance was accomplished although lower ammonium conversion ratio due to decreased activity of AOB and AMX. In addition, microbes assigned to genus 20

Thermomonas were capable of reducing nitrate in phenol wastewater under oxygen-limited conditions [28]. The denitrifying and bio-refractory organics-degradation microbes could eliminate part of carbon source, resulting in less inhibition on AOB and AMX.

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The experiment provided the information of microbial community structure and relative abundance variation during construction of hypothermic systems (Period I)

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and acclimatizing to raw leachate (Period II). The succession of microbial consortia implied that the nitrogen removal pathway shifted from PN/ANAMMOX to SNAD

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process as the substrate containing bio-refractory organics. The results were sugges-

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A

industrial wastewater under low temperature.

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tive for practical application of SNAD process for mature landfill and homogeneous

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4. Conclusions

This work provides perspective for efficient treatment of mature leachate at low

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temperature by SNAD. In Period I, the PN/ANAMMOX bioreactors fed with am-

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monia-rich wastewater were capable of acclimatizing to stepwise reduced temperature as low as 10 ℃ for prolonged operation (370 days), with average 79.7% and 78.4% nitrogen removed mainly ascribed to the consortia of AOB and AMX at 15 ℃

A

and 10 ℃, respectively. During Period II, the bioreactor turned out to be feasible for prolonged treatment of mature landfill leachate under 15 ℃ for 86 days, accomplishing simultaneous COD and nitrogen removal at 68.5% and 82.4%. The results of microbial succession implied that the SNAD process together with robust degrada21

tion of bio-refractory compounds occurred. Better nitrogen elimination performance during Period II might be ascribed to the reappearance of heterotrophic denitrifying bacteria (9.88%) and the complete suppression of NOB, in spite of 6.05% AOB and 4.56% AMX were inhibited by raw leachate. Moreover, the enriched bio-refractory

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organics degradation bacteria (24.38%) were conductive to the stabilization of sys-

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tem treating mature landfill leachate.

Acknowledgements

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This research has been carried out with the financially support from the National

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Science and Technology Major Project for Water Pollution Control & Remediation

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of China (Grant No. 2012ZX07307-002) and Graduate Scientific Research & Inno-

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vation Foundation of Chongqing (Grant No. CYB17005). The author would like to

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Table captions

Table 1. Characteristics of raw leachate in the study.

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Table 2. Nitrogen and COD removal rates during Period I and Period II

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Table 3. Bacteria diversity and richness index among sludge samples.

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Table 1. Characteristics of raw leachate in the study. Parameter

Value (mean ± S.D.) a

COD

1039 ± 45

BOD5

24.9 ± 7.4

NH+4 -N

1931 ± 47 26

<1

NO¯3 -N

<1

TKN

1949 ± 68

pH

8.4 ± 0.3

Values are in mg·L-1 except pH.

Table 2. Nitrogen and COD in the effluent during Period I and Period II Effluent (mg·L-1)

Period I 20 ℃

Period II a

15 ℃

a

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30 ℃

a

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a

NO¯2 -N

10 ℃

a

b

34.3±3.6

65.3±3.7

102.1±5.3

154.6±5.4

165.4±3.0

NO-2 -N

1.4±0.2

2.6±0.6

3.0±0.3

0.6±0.2

20.8±1.4

NO-3 -N

217.8±3.1

254.0±4.7

286.9±2.8

TN

253.5±2.4

321.9±4.1

391.0±4.4

COD

-

-

U

NH+4 -N

A

N

269.6±4.8

-

Influent nitrogen loads were 250 g N/(m3·d);

b

Influent nitrogen loads were 100 g N/(m3·d).

424.8±8.0

350.8±3.5

-

331.0±7.3

ED

M

a

164.6±2.8

Ace

1#

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Table 3. Bacteria diversity and richness indexes among sludge samples.

37306

144

156

161

0.999464

2.47

0.2079

2#

33399

157

169

172

0.999125

2.79

0.1254

3#

30645

161

177

176

0.999217

2.89

0.1068

4#

24388

127

120

122

0.999207

2.18

0.2265

5#

36462

278

287

288

0.999506

4.24

0.0247

Reads

OTU

Chao

Coverage Shannon Simpson

A

CC E

Sample

Figure captions Fig. 1. Schematic diagram and experimental procedure of the SBBR system. 27

Fig. 2. Evolution of (a) the nitrogen compounds concentration in effluent, (b) nitrogen removal efficiency during Period I. Point A, B, C represented that the influent loads were decreased to 188, 125 and 100 g N/(m3·d) on day 272, 316 and 344, respectively.

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Fig. 3. The revolution of (NH+4 -Ncon - NO¯2 -Nacc)/ NO¯3 -Npro as operation temperature decreased stepwise.

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Fig. 4. Evolution of nitrogen compounds concentration of the effluent (mg·L-1) in the SBBR during Period II. (a) Variation of COD concentration and COD removal ratio,

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(b) TN concentration and TN removal ratio, (c) ammonium and nitrate, (d) nitrite.

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Fig. 5. Principal component analyses (PCA) of samples based on microbial consortia.

A

Fig. 6. Sequencing assignment results among samples at the (a) phylum level, (b) ge-

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nus level. Only relative abundance count over 0.5% was shown in this figure.

A

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ED

Fig. 7. Succession of functional microbes for nitrogen and COD conversion.

28

IP T SC R U N A M

A

CC E

PT

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Fig. 1. Schematic diagram and experimental procedure of the SBBR system.

29

IP T SC R U N A M

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Fig. 2. Evolution of (a) the nitrogen compounds concentration in effluent, (b) nitrogen removal efficiency during Period I. Point A, B, C represented that the influent

A

CC E

spectively.

PT

loads were decreased to 188, 125 and 100 g N/(m3·d) on day 272, 316 and 344, re-

30

IP T SC R U N

A

Fig. 3. The revolution of (NH+4 -Ncon - NO¯2 -Nacc)/ NO¯3 -Npro as operation temperature

A

CC E

PT

ED

M

decreased stepwise.

31

IP T SC R U N A M ED PT CC E

Fig. 4. Evolution of nitrogen compounds concentration of the effluent (mg·L-1) in the

A

SBBR during Period II. (a) Variation of COD concentration and COD removal ratio, (b) TN concentration and TN removal ratio, (c) ammonium and nitrate, (d) nitrite.

32

IP T SC R U N

A

CC E

PT

ED

M

A

Fig. 5. Principal component analyses (PCA) of samples based on microbial consortia.

33

IP T SC R U N A M ED PT CC E A Fig. 6. Sequencing assignment results among samples at the (a) phylum level, (b) genus level. Only relative abundance count over 0.5% was shown in this figure. 34

IP T SC R U N

A

CC E

PT

ED

M

A

Fig. 7. Succession of functional microbes for nitrogen and COD conversion.

35