NOB suppression and adaptation strategies in the partial nitrification–Anammox process for a poultry manure anaerobic digester

Process Biochemistry 58 (2017) 258–265

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NOB suppression and adaptation strategies in the partial nitrification–Anammox process for a poultry manure anaerobic digester

MARK

Constanza Arriagadaa, Víctor Guzmán-Fierroa, Elisa Giustinianovicha, Luz Alejo-Alvareza, ⁎ Jack Behara, Luis Pereirab, Víctor Camposb, Katherina Fernándeza, Marlene Roeckela, a

Bioengineering Laboratory, Department of Chemical Engineering, Faculty of Engineering, University of Concepción, P.O. Box 160-C, Correo 3, Concepción, Chile Environmental Microbiology Laboratory, Department of Microbiology, Faculty of Biological Sciences, University of Concepción, P.O. Box 160-C, Correo 3, Concepción, Chile b

A R T I C L E I N F O

A B S T R A C T

Keywords: Organic matter adaptation Anammox Partial nitrification SNAD NOB

Poultry manure contains high levels of ammonia, which result in a suboptimal bioconversion to methane in anaerobic digesters (AD). A simultaneous process of nitrification, Anammox and denitrification (SNAD) in a continuous granular bubble column reactor to treat the anaerobically digested poultry manure was implemented. Thus, two strategies to achieve high efficiencies were proposed in this study: (1) ammonia overload to suppress nitrite oxidizing bacteria (NOB) and (2) gradual adaptation of the partial nitrification–Anammox (PN–A) biomass to organic matter. During the NOB-suppression stage, microbial and physical biomass characterizations were performed and the NOB abundance decreased from 31.3% to 3.3%. During the adaptation stage, with a nitrogen loading rate of 0.34 g L−1 d−1, a hydraulic retention time of 1.24 d and an influent COD/N ratio of 2.63 ± 0.02, a maximum ammonia and total nitrogen removal of 100% and 91.68% were achieved, respectively. The relative abundances of the aerobic and the anaerobic ammonia-oxidizing bacteria were greater than 35% and 40% respectively, during the study. These strategies provided useful design tools for the efficient removal of nitrogen species in the presence of organic matter.

1. Introduction Poultry manure can cause a serious environmental damage by polluting water and air, which can become harmful to both human and animal health [1]. Anaerobic digestion (AD) of these wastes is considered the best alternative to minimize the amount of poultry manure and to recover energy via the production of methane. This anaerobic digestion is a promising substitute for fossil fuels and has significant advantages over other forms of bioenergy production [1]. The major problem with AD is that poultry manure contains a high concentration of ammoniacal species (ammonium + ammonia), which result in a suboptimal production of methane [2]. Free ammonia (NH3) inhibits the methanogenic process by increasing the maintenance energy requirement, affecting the intracellular pH, depleting the intracellular potassium and inhibiting specific enzymatic reactions, mainly of archaea populations [3]. Ammonium inhibition at industrial scale leads to serious economic and operational problems in the biogas production process. In fact, many full-scale anaerobic digesters are operating in an ammonia-induced “inhibited steady-state”, with up to 30% losses of potential methane production yield [2]. In addition, anaerobic reactors operated with high ammonia concentrations generate an effluent

⁎

with a high concentration of ammonia that requires further treatment. The addition of a nitrogen treatment step over the anaerobic digester effluent would significantly reduce the ammonia contained in these wastewaters, which can be used to dilute the input stream, thereby reducing the ammonia load and avoiding methanogenesis inhibition [4]. To date, many strategies for removing ammonia have been developed, highlighted by autotrophic removal based on partial nitrification–Anammox reactions (PN–A), because it is not necessary to add organic matter. In addition, this process has lower oxygen requirements than traditional methods based on nitrification–denitrification reactions [5–7]. The partial nitrification is carried out by aerobic ammonia oxidizing bacteria (aerAOB), which oxidizes ammonia to nitrite whereas the Anammox reaction consists of the anaerobic oxidation of ammonia using nitrite as electron acceptor, carried out by anaerobic ammonia oxidizing bacteria (anAOB). According to the global reaction (Eq. (I)) described by Sliekers et al. [8], 89% of nitrogen can be removed as N2 and 11% remains as nitrate. NH3 + 0.85 O2 → 0.11 NO3− + 0.44 N2 + 0.14 H+ + 1.43 H2O

(I)

Autotrophic nitrogen removal technology has been successfully implemented in more than 100 plants worldwide; where most of them

Corresponding author. E-mail address: [email protected] (M. Roeckel).

http://dx.doi.org/10.1016/j.procbio.2017.03.028 Received 9 November 2016; Received in revised form 2 March 2017; Accepted 26 March 2017 Available online 11 April 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.

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considering the typical low nitrite concentration found in the bulk of a PN–A reactor and the kinetic parameters such as nitrite saturation coefficient and the maximum growth rate of NOB, if NOB activity is developed; both growth and the activities of denitrifying bacteria and especially of anAOB are compromised [18,19]. Thus, the design of a NOB suppression strategy prior to the adaptation of the PN–A biomass to organic matter in wastewaters with organic matter using PN–Anammox process becomes imperative. The coupled PN–A reactions produce 11% of residual nitrogen in the form of nitrate, due to the stoichiometry reaction. Thus, in the presence of organic carbon, the remaining nitrate can be used by denitrifying bacteria as electron acceptors, improving the N removal efficiency. This new process has been called the simultaneous nitrification, Anammox and denitrification (SNAD) process [10]. It is also relevant to consider the assimilation of nitrogen due to the proliferation of heterotrophic biomass, which improves the performance of the system [20]. In this study, strategies for a start-up with NOB suppression and for the adaptation of the PN–A process to a SNAD process have been proposed. The effects of digested poultry manure as a substrate for the SNAD process were evaluated in a continuously fed granular bubble column reactor (BCR). The effects of diluted effluent from the anaerobic digester on the PN–A reactor performance and on the biomass quality, such as specific biomass activities, granule diameter, sedimentation properties and microbial composition, were also evaluated. Fig. 1. Scheme of PN–A reactor. (1) Gas trap. (2) Effluent stream. (3) Feeding stream. (4) Heating water output. (5) Heating water input. (6) Gas recirculation. (7) Dissolved oxygen [DO] measurement. (8) Air make-up. (9) Inlet air flow. XT, DO transmitter; XC, DO controller; CM, control module.

2. Materials and methods 2.1. Experimental unit

treat the supernatant of anaerobic sludge digesters [9]. However, in the case of anaerobic digesters that treat industrial effluents, the presence of certain amounts of chemical oxygen demand (COD) is common [10,11]. The presence of organic carbon can lead to an imbalance of the N removal process due to the competition between heterotrophic and autotrophic bacteria because heterotrophs have a higher growth rate, easily outcompeting autotrophic bacteria for substrates (such as nitrite and oxygen) and for living space [11,12]. Consequently, the development of a multiparametric strategy to prevent heterotrophic biomass contamination becomes necessary. In addition, nitrite oxidizing bacteria (NOB) competes for oxygen with aerAOB as well as for nitrite with anAOB; thus, if the growth of NOB is not prevented, the nitrate production is increased whereas the efficiency of the process is decreased [13]. Therefore, the oxidation of nitrite to nitrate by NOB is undesirable and it should be avoided. The NOB inhibition in a PN–A process has been widely researched. There are different registered technologies based on the PN–A process that consider several strategies such as: controls of sludge retention time (SRT), pH, dissolved oxygen limitation, aeration intensity, redox potential and concentrations of free ammonia [8,14–16]. Varas et al. [17] reported 75.36% of total nitrogen removal with an oxygen limitation strategy. However, molecular analyses have demonstrated that the NOB group was the most abundant bacteria. Hence, the oxygen limitation promoted NOB inhibition without NOB suppression. Moreover, during the operation of the PN–A process with organic matter the nitrite is a crucial limitation, because it can be consumed by NOB, an AOB as well as by denitrifying bacteria. By

A continuously fed BCR with a working volume of 4.3 L was started and used. The BCR was chosen because in a previous study by our working group, Varas et al. [17] obtained good performances through a continuously fed BCR with a stable operation reaching around 75% of total nitrogen removal. The reactor had two sections: the lower section had an inner diameter and height of 7.0 and 75.0 cm respectively, whereas the upper section, whose function is to reduce the ascending speed of the granular biomass, had an inner diameter and height of 9.5 and 40.0 cm respectively. In the upper section, a three-phase separator in which the separation between the granular biomass and the flocculent biomass occurred was integrated. The separator consisted in three concentric pieces (two cones and one cylinder) placed one over the other with 1 cm of separation. From bottom to top, the allocation is as follows: both cones and the cylinder. The inlet and outlet streams were controlled with a two-head peristaltic pump. Oxygen control system was performed according to the strategy proposed by Varas et al. [17]. The PN–A reactor is shown in Fig. 1. 2.2. Start-up and operation strategy The PN–A reactor operated at a constant temperature of 35 °C, a pH between 7.5 and 8.0, and a constant oxygen concentration of 0.2 and 1.0 mg O2 L−1 (depending on the evaluation stage, Table 1) was used; where oxygen was the only controlled parameter. The inoculum biomass corresponded to a preacclimated cell suspension from a

Table 1 Operational stages of the PN–A reactor during the stabilization period. Stages

Days

TAN influent (mg N L−1)

HRT (d)

pH

NLR (g TAN-N L−1 d−1)

Oxygen concentration (mg O2 L−1)

Objectives

I

1–57

120–570

2.44

6.43–8.37

0.05–0.23

0.2

II-a II-b III

58–84 85–91 91–202

550–200 200–400 400

2.44 2.44 2.44–1.24

7.47–8.34 7.32–8.05 6.68–8.21

0.23–0.08 0.08–0.16 0.16–0.32

0.2 1.0 0.2

NOB suppression by free ammonia overload aerAOB activity recovery aerAOB growth anAOB activity recovery

TAN, total ammonia nitrogen; HRT, hydraulic retention time; NLR, nitrogen loading rate.

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Table 2 Operational stages of PN–A reactor during the organic matter adaptation period. Stagesa

Days

TAN Influent (mg N L−1)

pH

Objectivesb

TNRc

ANRd

IV V VI VII

203–230 231–247 248–265 266–280

278–545 364–556 334–439 392–439

7.36–8.16 7.38–7.73 7.19–7.53 6.69–7.90

25% diluted effluent from the anaerobic digester 50% diluted effluent from the anaerobic digester 75% diluted effluent from the anaerobic digester 100% diluted effluent from the anaerobic digester

80.68% 74.29% 75.53% 83.22%

95.15% 91.69% 90.47% 89.85%

a b c d

Hydraulic retention time, HRT = 1.24 d; and dissolved oxygen, DO = 0.2 mg O2 L−1 during all stages. Mixed with synthetic substrate described in Section 2.3 and diluted to approximately 400 mg N L−1. Total nitrogen removal (mean). Ammonia nitrogen removal (mean).

2.4.1. Relative abundance of bacterial The fluorescence in situ hybridization (FISH) technique was used to determine the relative abundance of the bacterial consortium in the granular biomass. Both preparation and visualization of the samples were carried out as reported by Varas et al. [17]. AnAOB (Anammox), aerAOB and NOB were hybridized with the probes AMX820, NEU653 and Ntspa 1026, respectively. DAPI and EUB-338 (eubacteria) were used as controls. The probes were selected according to Cui [13] and they are included in Supplementary data section (Table S1). The domain-specific probe EUB-338 was used to determine the eubacteria population and the analysis was conducted in a MOTIC-BA310 fluorescence microscope, whereas the imaging was performed using the Motic Images Plus 2.0 digital camera with 100× immersion objective. For quantification of the images 10 fields randomly taken were analyzed, the image analysis was performed through the image processor Motic Images Plus 2.0 software. The difference between eubacteria and the sum of each bacterial group was called unidentified eubacteria (EUBUI). Then, the percentage of the relative abundance corresponded to the ratio between each bacterial group (anAOB, aerAOB, EUBUI and NOB) and eubacteria.

PN–A reactor with synthetic substrate, previously described by Varas et al. [17]. The initial biomass concentration was 0.55 g VSS L−1. The operation was divided into two periods: stabilization and adaptation (Tables 1 and 2, respectively). During the stabilization period, the reactor was fed only with the synthetic substrate, and during the adaptation period, the synthetic substrate was gradually replaced with the digester liquor of poultry manure. 2.3. Substrate Initially, the reactor was operated with the synthetic media, as described by Varas et al. [17]. During the adaptation of the biomass, a mixture of synthetic and real substrates was used. The real substrate was a supernatant obtained from decanted effluent of a poultry manure anaerobic digester (Table 3), diluted to achieve a concentration of 400 mg TAN-N L−1. The adaptation was performed in four stages in which the diluted effluent from the anaerobic digester (dilution factor = 3.5) replaced the synthetic substrate as follows: 25%, 50%, 75% and 100% (v/v) (Table 2).

2.4.2. Aerobic and anoxic batch activities tests Respirometric batch experiments were performed to measure the specific nitrification activity (SNA) with aerobic batch activity tests according to the procedure described by Dapena-Mora et al. [21]. A total of 4 mL of granules were obtained and washed 3 times with phosphate buffer (KH2PO4 0.14 g L−1 and K2HPO4·3H2O 0.9824 g L−1). The test was previously validated considering the accuracy of the oxygen probes and the concentrations of biomass. The biomass and buffer were placed in a jar with a final volume of 449 mL and incubated with an oxygen electrode at 35 °C with magnetic stirring. The medium was saturated with air for 10 min. Subsequently, the flask was closed and allowed to react to observe endogenous oxygen uptake for another 10 min; next, 1 mL of substrate (38.16 g TAN-N L−1) was injected and was allowed to react for the same amount of time. During this period, the total oxygen consumption by the biomass was observed. After each test, volatile suspended solids (VSS) were measured using the standardized method, allowing the activity to be expressed as SNA (g N-NH4+ g VSS−1 d−1). Specific Anammox activity (SAA) was measured based on increased pressure using OxiTop® Control AN6 (WTW, Weilheim, Germany) according to Varas et al. [17].

2.4. Physical and biological characterization of the granules Both physical and biological characterizations of the granules were performed during all the stages of the adaptation period to the diluted effluent from the anaerobic digester, in the PN–A reactor. In this study we focused on the characteristics of the granular biomass, as it contains the slower growth biomass fractions and its changes better represent the long-term adaptation of the biomass in the reactor. At the same time, the flocculent biomass can change more easily and quickly with fluctuations in the medium, and it is also affected to changes in the characteristics of the separator, as we observed during the adaptation stage. Therefore, the behavior of the reactor in a long term is not represented only by the flocculent biomass. Table 3 Characterization of the anaerobic digester liquor of poultry manure used as the substrate for the PN–A reactor.

TSS VSS COD TOC NO2−-N NO3−-N TAN-N TKN

g SST 100 mL−1 of digestate g SSV 100 mL−1 of digestate (mg O2 L−1) (mg C L−1 sample) (mg N L−1 sample) (mg N L−1 sample) (mg N L−1 sample) (g N L−1 sample)

Mean ± SD

Minimum

Maximum

0.4 ± 0.04

0.37

0.45

0.2 ± 0.03

0.18

0.25

2775.7 ± 274.0 730.5 ± 95.8 0.00 5.4 ± 7.6 1403.8 ± 384.6 1960.6 ± 565.9

2522 651 0 11 1020 1202

3066 869 0 24 1869 2455

2.4.3. Sedimentation properties and granule measurements The sedimentation rate of 30 granules was determined by measuring the time required for each granule to travel 30 cm in a transparent vertical column (diameter of 10 cm) filled with water (20 °C), as described by Vlaeminck et al. [22]. The sludge volumetric index (SVI) was determined by measuring the volume occupied by the biomass contained in a test tube after 30 min of sedimentation, as described by Dapena-Mora et al. [21]. The equivalent granule diameter (da) was measured with a representative image of a sample containing 100 granules according to the

TSS, total suspended solids; VSS, volatile suspended solids; COD, chemical oxygen demand; TOC, total organic carbon; TAN-N, total ammonia nitrogen; TKN, total Kjeldahl nitrogen.

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aerAOB growth and, finally, an AOB activity recovery by decreasing DO (Table 1). The initial strategy (Stage I, Table 1) of NOB suppression by free ammonia overload was performed. The PN–A inoculum was harvested from another PN–A reactor with a nitrogen removal around 75%, though with a NOB biomass abundance of 32.1% [17]. For this reason, the achievement of NOB suppression and the avoidance of oxygen competition with aerAOB were essential. Varas et al. [17] operated a continuously fed granular BCR with different oxygen concentrations in which 0.2 mg O2/L was the best condition. Therefore, a strategy with oxygen as the only limiting factor was discarded, and NOB suppression by free ammonia overload was selected. During this stage (Stage I, Table 1), the initial nitrogen loading rate (NLR) in the influent was 0.05 g TAN-N L−1 d−1; then, the TAN-N concentration was increased until reaching an NLR of 0.23 g TAN-N L−1 d−1. This increase was through TAN concentration of the influent. Commonly, NOB is more sensitive to free ammonia (NH3) than aerAOB, and different tolerance limits have been detected. Anthonisen et al. [25] and Vadivelu et al. [26] observed NOB inhibition at NH3 concentrations below 1 mg L−1 whereas aerAOB showed inhibition above 16 mg L−1. At the 57th day of operation (Fig. 2), when the free ammonia concentration was approximately 50 mg NH3-N L−1 and DO concentration was 0.2 mg O2/L, the ammonia and total nitrogen removal decreased from 90% to 53% and from 76% to 46%, respectively. Nevertheless, the NOB abundance decreased a 90% at the end of the stabilization period (Fig. 2). Then, NOB suppression was achieved. The second stage (Stage II, Table 1) was focused on the recovery of the aerAOB activity and promoting aerAOB growth. First (Stage II-a, Table 1), the NLR was gradually decreased from 0.23 to 0.08 g TANN L−1 d−1 (through TAN influent concentration decrease) without successful results. The removal of ammonia and total nitrogen during this period was approximately 52.20% and 39.61%, respectively, due to the strong inhibition caused at Stage I. Second (Stage II-b, Table 1), a high DO concentration was set at 1.0 mg L−1 and TAN influent concentration was increased in order to promote growth and prevent underfeeding of aerAOB, consequently providing anAOB protection from oxygen inhibition by recovering the granules with an aerAOB layer. Before the increment of both parameters, TAN concentration in the bulk liquid was around 66 mg TAN L−1, and the increase of TAN influent and oxygen concentration as a strategy was also confirmed. As a consequence of those changes, the TAN effluent concentration was 0 mg TAN L−1 at the end of Stage II-b (Fig. 2). Results have revealed an increase in ammonium oxidizing activity, achieving 100% ammonia removal and low NOB activity by the nitrite accumulation. Nitrogen removal activity decreased, as expected, until reaching 6.90% because of oxygen inhibition of anAOB due to the high DO concentration. Once the ammonia removal reached 100%, Stage II-b finished, avoiding the increase of NOB activity. In addition, the reactor was also diluted to prevent an irreversible inhibition of anAOB because of the high nitrite concentration reached, 250 mg NO2−-N L−1. Finally, the third stage (Stage III, Table 1) was focused on restoring the anAOB activity and achieving a steady state in the PN–A reactor. A low DO concentration of 0.2 mg L−1 was fixed in the reactor, and while recovering the anAOB activity, the NLR was increased from 0.08 to 0.16 g TAN-N L−1 d−1 by decreasing the HRT. A stable operation was achieved at day 139, with an NLR of 0.323 g N L−1 d−1 (Fig. 2), obtaining ammonia and total nitrogen removals of approximately 89.2% and 79.9%, respectively. A reactor activity of 0.17 g N g VSS−1 d−1 was achieved, which was slightly higher than the removal and activity reported by Varas et al. [17] under similar operating conditions, indicating that this result is attributed to the NOB suppression. In addition, the FISH analysis corroborated a strong decrease of the NOB group in the granular biomass and at the end of the stabilization period the percentage of the relative abundance of aerAOB, anAOB, NOB and EUBUI was 37.88, 40.67, 3.34 and 18.11%, respectively. On

procedure reported by Varas et al. [17]. 2.5. Analytical methods Periodically, both inlet and outlet streams from the reactor were sampled. Nitrogen compounds such as nitrate, nitrite and total ammonia nitrogen (TAN-N) were measured in a flow injection analyzer (FIAlab, 2500/2700, 1.0607, Seattle, WA, USA) using a USB400-VISNIR detector according to the method proposed by Sánchez et al. [23]. Measurement of total suspended solids (TSS), VSS and COD was performed according to standard methods. The total Kjeldahl nitrogen (TKN) was measured in a distillation unit (Büchi 323, Switzerland) after a digestion step (Büchi, 435, Switzerland). The total organic carbon (TOC) was measured via combustion analysis followed by a nondispersive infrared gas analyzer in the TOC (Shimadzu, TOC-5000a, Japan) [24]. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey's test as a post hoc analysis with GraphPad Prism version 5.0 (GraphPad software, USA). P values < 0.05 were considered statistically significant. 2.6. Calculations The percentage of nitrogen removal was calculated to determine the efficiency of the process. The removal of ammonia nitrogen (ANR) and total nitrogen (TNR) were calculated through the expressions suggested by Varas et al. [17]. The calculations were performed through the following expressions:

⎛ TANoutlet ⎞ ANR=⎜1 − ⎟ × 100% TANinlet ⎠ ⎝

(1)

⎛ TANoutlet + NO3−Noutlet + NO2−Noutlet ⎞ TNR=⎜1 − ⎟ × 100% TANinlet + NO2−Ninlet ⎝ ⎠

(2)

−

where TANinlet and NO2 Ninlet are the TAN and nitrite concentrations in the influent stream to the reactor respectively. Inlet nitrate concentration was not considered because the system was fed with TAN and nitrite. TANoutlet, NO2−Noutlet and NO3−Noutlet are the TAN, nitrite and nitrate concentrations in the effluent stream of the reactor, respectively. The PN–A reactor activity was measured with a modification in the unit of measurement employed on the expression suggested by Varas et al. [17]:

A [gN/gVSS d] =

(Ninlet − Noutlet ) [gN/L] τ[d ]Cbiomass [gVSS/L]

(3)

where Ninlet and Noutlet are the nitrogen concentrations in the inflow and outflow streams of the reactor, respectively, τ is the hydraulic retention time (HRT), and Cbiomass is the biomass concentration measured as VSS. Free ammonia was calculated based on the equation suggested by Anthonisen et al. [25]. It was assumed that a steady state condition was reached when the measured efficiency remained nearly constant, i.e., when no difference larger than 5% was observed for different days. 3. Results and discussion 3.1. Stabilization of the PN–A reactor The autotrophic removal of nitrogen in a PN–A reactor was studied in two different periods: stabilization and adaptation (Tables 1 and 2, respectively). In order to achieve a robust PN–A process capable of tolerating the addition of organic matter, a stabilization strategy was implemented. The strategy consisted of three stages: NOB suppression by free ammonia overload, recovery of the aerAOB activity and promotion of 261

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Fig. 2. Profile during the adaptation of the PN–A reactor. (A) Inlet and outlet nitrogen species. (B) Ammonia nitrogen removal (ANR) and total nitrogen removal (TNR). Operating conditions: reactor volume = 4.3 L and T = 35 °C.

Fig. 3. Microbiological characterization of the granular PN–A inoculum biomass and PN–A biomass during different stages of adaptation. Ammonia-oxidizing bacteria (aerAOB), Anammox (anAOB), nitrite-oxidizing bacteria (NOB) and unidentified eubacteria (EUBUI).

biomass through the control of the SRT with a slight modification of the separation system. The proposed control must to be gradual in order to maintain a denitrifying activity on the reactor, a situation that is prevented in the rest of the systems [8]. Nevertheless, the growth of denitrifying bacteria over the granular biomass should be avoided in order to increase the process efficiency. During the study, when the biomass concentration on the reactor was 6.5 g VSS L−1, the SRT was 10 d. After the slight modification of the separation system, the biomass concentration on the reactor decreased to 3.5 g VSS L−1 and the SRT was 5.4 d. Moreover, the washed-out biomass mainly corresponded to flocs, achieving good granular biomass retention. The control of HRT and SRT allowed controlling the suppression of NOB biomass and the development of denitrifying biomass in the reactor. Thus, when working with high C/N ratio, the control of SRT, free ammonia concentration and oxygen concentration allow regulating the proliferation of NOB and denitrifying biomass and improve the performance of the reactor. Another important factor in the removal of nitrogen is ammonia assimilation by heterotrophic biomass. Thus, heterotrophic assimilation of nitrogen, control of denitrifying activity and suppression of NOB biomass allow the increase the nitrogen removal. Ammonia removals and total nitrogen removals obtained at each step are shown in Table 2. During stage VII (days 266–280), the PN–A reactor was fed with 100% volume of diluted effluent from the

the other hand, for the inoculum the relative abundances were 31.3, 17.4, 32.1 and 19.2%, respectively [17] (Fig. 3). These results revealed a decrease of nearly 90% of NOB abundance, corroborating the effectiveness of the start-up implemented strategy. These results contribute to the strategies described for the start-up of PN–A process [8,14–17], since so far it has been approached only the inhibition of NOB without consider a bioprospecting of the bacterial consortium in the biomass that will ensure the suppression of the undesirables species. Therefore, it can be concluded that one of the common problems in the PN–A reactor operation, where NOB becomes a contaminant and the other microorganisms will be not capable to compete with it, can be overcome by multiparametric strategies. 3.2. Adaptation of the PN–A reactor to organic matter After day 203, the adaptation period was performed in four steps, corresponding to different increasing ratios of 25, 50, 75 and 100% (v/ v) of diluted effluent from the anaerobic digester/total substrate. The diluted effluent from the anaerobic digester was the supernatant of decanted effluent of an anaerobic digester fed with poultry manure which was mixed with synthetic substrate, as described in Section 2.3. The aim of a gradual adaptation of the PN–A biomass to organic matter was to avoid an excessive growth of heterotrophic flocculent 262

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(Fig. 3). Summarizing, a greater efficiency was achieved when the adaptation was completed (100% (v/v) of diluted effluent from the anaerobic digester), and a maximum of 91.68% total nitrogen removal was reached with a COD/N ratio of 2.63 (organic load of 864 mg COD L−1 d−1). However, for high COD/N ratios, an effective biomass separation system in the SNAD reactor is essential to allow both the outflow of suspended biomass and the retention of granules. If the biomass retention system is not well designed, reactor clogging problems or the washing out of biomass cannot occur and the process will collapse.

anaerobic digester and at this stage, approximately 89.95% and 83.84% removal of ammonia and total nitrogen, respectively, was observed. As shown in Fig. 2B, the maximum ammonia and total nitrogen removal was obtained during the steady state of stage VII (days 275–280), achieving values of 98.46 ± 1.09% and 90.28 ± 1.55% respectively, and a reactor activity of 0.080 g N g VSS−1 d−1 was attained. During the entire adaptation period, the effluent quality was relatively constant, considering the typical affluent fluctuations from a digested. The high removal efficiencies obtained exceeded the stoichiometric quantities expected for a PN–A process. The coupled reactions of partial nitrification and Anammox are capable of removing a maximum of 89% of ammonium, leaving the remaining 11% of nitrogen as nitrate species. In the presence of organic matter, the remaining nitrate can be used by heterotrophic biomass as an electron acceptor for the oxidation of organic carbon, improving the N removal efficiency. The SNAD process [10] was observed in this study with high N removal efficiency (Fig. 2) and COD removal between 41.38% and 77.42% at the end of it. Another COD removal path could be the ability of anAOB to oxidize volatile fatty acids, which are produced during the acidogenic stage in the AD, using nitrate or nitrite as an electron acceptor [27,28] without incorporating the fatty acids in the biomass; however, this process completely converts them to carbon dioxide gas (CO2) [27]. Nevertheless, heterotrophic bacteria (HB) were well developed and strongly competed with anAOB for organic matter. Competition phenomena between heterotrophs and Anammox bacteria for electron acceptors and living space are well documented [29–31]; in addition, in PN–A systems, inhibition problems occurred at COD/N ratios even lower than 1, reducing the N removal efficiency [10,12]. Compared to other PN–A results in the presence of organic matter and considering the high COD/N ratio used for a PN–A process (2.63 ± 0.02), results of the present study are among the most successful. Wang et al. [32] and Joss et al. [33] obtained a maximum of 94% of nitrogen removal in PN–A aeration tanks, working with landfill leachate (COD/N ratio of 0.85) and the digested supernatant of a municipal wastewater treatment plant (COD/N ratio of 0.5), respectively. Daverey et al. [34] obtained 80% of nitrogen removal efficiency in a granular aeration tank fed with anaerobic digester liquor of swine wastewater with a variable COD/N ratio of approximately one. In this study, a nitrogen removal efficiency of 90.28 ± 1.55% was achieved with the effluent of a poultry manure anaerobic digester (diluted) as the substrate, with a high COD/N ratio of 2.63 ± 0.02. Moreover, at days 278 and 280, when the total nitrogen removal was 90.42% and 91.68% it was reached a COD removal of 41.38% and 77.42%, respectively. Furthermore, if COD removal is due to the presence of heterotrophic biomass, then nitrogen was also removed by bacterial assimilation. For example, the yield of heterotrophic biomass is 0.43 g VSS/g COD and the assimilation of ammonia by heterotrophic bacteria can be around 10% of the biomass weight (0.1 g N/g VSS) [20] therefore, the ratio of assimilated nitrogen to COD removed is 0.043 g N/g COD. Now, if the inlet COD was 1071 mg COD L−1 and around 41.38–77.42% of COD was removed, the ammonia removal from assimilation can be 19–35 mg N L−1. Then, ammonia assimilation by heterotrophs is an important fact for the nitrogen removal of the reactor. The process implemented in this study presents a high nitrogen removal due to the combination of the two strategies: NOB suppression and the control of the flocculent biomass SRT. It is well known that the suspended biomass can grow if HRT is higher than the inverse of the maximum specific growth rate of the microorganisms [35,36]. In this study, the HRT was set at values that avoided the wash out of suspended heterotrophic biomass (≥1.24 d), thus preventing the development of a heterotrophic layer over PN–A granules. During the adaptation period, the HRT was maintained at 1.24 d, allowing the suspended growth for heterotrophic biomass. The success of this strategy was corroborated by the high removal efficiency obtained and the stability of the granule communities, which were unaltered in their microbial composition during the adaptation process

3.3. Organic matter effect over PN–A granular biomass In order to study the effect of the diluted effluent from the anaerobic digester on the biomass, granular biomass of the PN–A reactor was physicochemically and microbiologically characterized. Changes in the biomass during were determined through the following processes: specific nitrification and Anammox activity assays (SNA and SAA, respectively), sludge volumetric index (SVI), granular sedimentation rate (GSR), equivalent granule diameter (da), and FISH analysis. The relative abundance of aerAOB (ANOVA, P = 0.9437), anAOB (Anammox) (ANOVA, P = 0.9822), NOB (ANOVA, P = 0.1177) and EUBUI (ANOVA, P = 0.853) of the granular biomass during the adaptation period were not significantly different within a 95% confidence interval, as shown in Fig. 3. The sum of anAOB and aerAOB was greater than 75% for all stages, whereas the percentage of NOB remained at low values, between 1.87 and 4.60%. These results demonstrate the stability of the granular biomass under the studied conditions. Many authors have reported the existence of aerAOB and anAOB in PN–A systems in the presence of organic matter, and they related their abundance to the performance of the process [10,37,38]. However, few studies have evaluated the stability of granules, considering that the instability of this functional unit is one of the main problems of aerobic granulation technology [39]. Zhang et al. [12] observed a decrease in anAOB abundance due to a substrate change from synthetic to swine digester liquor and at increasing COD/N ratios over the range of 0.65–1.24. In this study, a stabilized granule biomass was achieved, which was because the HRT was greater than the maximum specific growth rate of heterotrophs [35] and because of the increase of organic load and limiting substrate (oxygen) [40]. Both conditions established the optimal conditions for the development of free-living biomass independent of aggregate biomass. Fig. 4 shows the results obtained for SNA and SAA at the end of the adaptation period, the SNA doubled, whereas the SAA was half the initial activity values. The SNA was the most variable parameter during the adaptation, reaching a peak of 1.272 g N-NH4+ g SSV−1 d−1 in the middle of the period (50% of diluted effluent from the anaerobic digester). An increase of NOB activity can explain the increase on SNA and the transient nitrate accumulation in the reactor (Fig. 2), with the consequent reduction of nitrogen removal. Because the microbial composition of the granular biomass was unaltered, it was assumed that the increase of SNA was due to an increase of suspended biomass which was revealed with the increase of SVI. The biomass augmentation inside the reactor, measured as VSS, increased from 1.5 g VSS L−1 at the beginning of the adaption to 6.5 g VSS L−1 in the middle of the period. Therefore, the separation system was slightly modified, allowing for a greater outflow of suspended biomass. Subsequently, the VSS decreased to 3.5 g VSS L−1 and the SNA also decreased; the nitrogen removal gradually increased, as shown in Fig. 4. Meanwhile, the SAA decreased from 0.41 g N-N2 g VSS−1 d−1 to 0.23 g N-N2 g VSS−1 d−1 at the end of the adaptation period. This range of values is within those reported by other authors with values between 0.10 and 0.53 g N-N2 g VSS−1 d−1 [10,41,42]; the range depends on the different operating conditions performed for each experiment. Fig. 5 shows the tendencies of both SVI and GSR during the 263

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Fig. 4. Characterization of the granular PN–A biomass during different stages of adaptation. Specific nitrification activity (SNA), specific Anammox activity (SAA) and total nitrogen removal (TNR) during the steady state.

Fig. 5. Physicochemical characterization of granular PN–A biomass during different stages of adaptation. Sludge volumetric index (SVI) and granular sedimentation rate (GSR).

suspended biomass and the granules retention. These strategies can be employed in industrial start up to reach a complete nitrogen removal in the presence of organic matter for protein-rich effluents, such as cattle, fish or other agriculture-based industry residues.

adaptation. The SVI showed an increase of 61.64% at the end of the adaptation period. However, the GSR showed no significant differences within a 95% confidence interval (ANOVA, P = 0.8737). Because SVI considers all biomass in the reactor whereas the GSR only considers the granular biomass, the increase in SVI was attributed to the increase of the suspended biomass in the reactor. This result is consistent with the increase in VSS and the unaltered microbial composition of the granular biomass. In addition, the equivalent granule diameter (da) and size distribution of the granules showed no significant differences within a 95% confidence interval (ANOVA, P = 0.9724), with a mean of 1.01 mm. Consequently, granular composition, size and properties were not affected by the organic substrate, and the operating conditions allowed the growth of suspended biomass without disturbing the granule properties. Thus, the results obtained from the SVI, the biomass sedimentation rate, granular diameter and the microbiological characterization (quantified as the relative abundance determined by FISH) confirmed the biomass stability during the adaptation period.

Acknowledgments This study was made possible by FONDECYT (Chile) [Grant Number 1140491] and INNOVA (Chile) [Grant Number 12IDL2-13605]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2017.03.028. References [1] F. Abouelenien, Y. Namba, M.R. Kosseva, N. Nishio, Y. Nakashimada, Enhancement of methane production from co-digestion of chicken manure with agricultural wastes, Bioresour. Technol. 159 (2014) 80–87, http://dx.doi.org/10.1016/j. biortech.2014.02.050. [2] I.A. Fotidis, H. Wang, N.R. Fiedel, G. Luo, D.B. Karakashev, I. Angelidaki, Bioaugmentation as a solution to increase methane production from an ammoniarich substrate, Environ. Sci. Technol. 48 (2014) 7669–7676, http://dx.doi.org/10. 1021/es5017075. [3] Y. Chen, J. Cheng, K. Creamer, Inhibition of anaerobic digestion process: a review, Bioresour. Technol. 99 (2008) 4044–4064, http://dx.doi.org/10.1016/j.biortech. 2007.01.057. [4] L. Alejo-Alvarez, V. Guzmán-Fierro, K. Fernández, M. Roeckel, Technical and economical optimization of a full-scale poultry manure treatment process: total ammonia nitrogen balance, Environ. Technol. 37 (2016) 2865–2878, http://dx.doi. org/10.1080/09593330.2016.1167963. [5] S. Vlaeminck, H. De Clippeleir, W. Verstraete, Microbial resource management of one-stage partial nitritation/anammox, Microb. Biotechnol. 5 (2012) 433–448, http://dx.doi.org/10.1111/j.1751-7915.2012.00341.x. [6] Y.H. Ahn, Sustainable nitrogen elimination biotechnologies: a review, Process Biochem. 41 (2006) 1709–1721, http://dx.doi.org/10.1016/j.procbio.2006.03. 033. [7] L.Y. Zhang, Y.B. Ye, L.J. Wang, B.D. Xi, H.Q. Wang, Y. Li, Nitrogen removal processes in deep subsurface wastewater infiltration systems, Ecol. Eng. 77 (2015)

4. Conclusions The feasibility and effectiveness of the SNAD continuous process adapted to digested poultry manure with a COD/N ratio greater than 2 were studied. A successful NOB-suppression stage was achieved, with a decrease of the NOB abundance from 31.3% to 3.3%. Because NOB competes for oxygen with aerAOB and for nitrite with anAOB, a multiparametric NOB suppression strategy design prior to the PN–A organic matter adaptation is crucial for successful nitrogen removal. With high COD/N ratios, an effective organic matter adaptation strategy was developed with 90.28 ± 1.55% of nitrogen removal at the steady state with 100% (v/v) of diluted effluent from the anaerobic digester. During a given adaptation period, the key factors to avoid competition between heterotrophic and autotrophic bacteria are the gradual increase of diluted effluent from the anaerobic digester/total substrate ratios, the prevention of the development of a heterotrophic layer over the PN–A granules by appropriate HRT, the outflow of 264

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