Nitrogen removal by simultaneous partial nitrification, anammox and denitrification (SNAD) in a structured-bed reactor treating animal feed processing wastewater: Inhibitory effects and bacterial community

International Biodeterioration & Biodegradation 133 (2018) 108–115

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Nitrogen removal by simultaneous partial nitrification, anammox and denitrification (SNAD) in a structured-bed reactor treating animal feed processing wastewater: Inhibitory effects and bacterial community

T

Ricardo Gabriel Bandeira de Almeidaa, Carla Eloísa Diniz dos Santosa,∗, Taíssa Colucio Lüdersa, Valéria Del Nerya, Cintia Dutra Lealb, Alyne Duarte Pereirab, Juliana Calábria Araújob, Russel J. Davenportc, Ana Cláudia Baranad, Deize Dias Lopese, Márcia Helena Rissato Zamariolli Damianovica a

Laboratory of Biological Processes, Centre for Research, Development and Innovations in Environmental Engineering, São Carlos School of Engineering, University of São Paulo, Av. João Dagnone 1100, 13563-120, São Carlos, SP, Brazil b Department of Sanitary and Environmental Engineering, Federal University of Minas Gerais (UFMG), Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil c School of Civil Engineering & Geosciences, Newcastle University, NE1 7RU Newcastle Upon Tyne, UK d Department of Food Engineering, State University of Ponta Grossa, Av. Gal. Carlos Cavalcanti, 4748, 84030-900, Ponta Grossa, PR, Brazil e Department of Civil Engineering, State University of Londrina, Rod. Celso Garcia Cid, Km 380, 86051-991, Londrina, PR, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Low C/N ratio Ion torrent sequencing Denitrification inhibition Free ammonia (FA) SNAD process

The aim of this study was to investigate the post-treatment of UASB effluent by treating animal feed production wastewater using simultaneous partial nitrification, anammox and denitrification (SNAD) in a structured-bed reactor subjected to low aeration and recirculation. The average nitrogen loads applied were 0.307, 0.249 and 0.149 kgN m−3d−1, correlated to COD/N ratios of 0.28, 0.41 and 0.26 (Phases 1, 2 and 3, respectively). The best mean values for removal efficiencies of total-N and COD were obtained in Phase 1 with 48 ± 24% and 63 ± 20%, respectively, reaching a maximum total-N removal efficiency of 79%. The anammox process was the main pathway of nitrogen removal, as pointed out in the nitrogen mass balance. High free ammonia (FA) concentrations in Phases 2 and 3, associated to the limitation of oxygen supply, caused inhibition of nitrite oxidizing bacteria (NOB) activity, leading to NO2 accumulation, having an impact on the denitrifying activity. At the end of the operational period, sequencing analysis detected sequences related to heterotrophic denitrifiers (22.5%), anammox bacteria, Candidatus Anammoximicrobium (2%) and ammonia oxidizing bacteria (AOB) belonging to Nitrosomonadales and Nitrosomonas (0.6%). These results demonstrated that nitritation, denitrification and anammox were likely in the processes involved in nitrogen removal in this reactor.

1. Introduction Pet food industries play an important role in preserving the environment and valuing agro-industrial waste. These companies use effluent produced in poultry and cattle slaughterhouses as raw material. If this waste was not reused, it would be disposed of in the environment (Jayathilakan et al., 2012; Wosiack et al., 2015). Effluent from poultry and cattle slaughterhouses has a high concentration of oils and grease, nitrogenous and sulfurous compounds derived from the protein breakdown (Jeganathan et al., 2006; Liu et al., 2004; Wosiack et al., 2015). This composition makes biological treatments difficult due to

∗

the complexity and high pollutant loading. According to Jeganathan et al. (2006), in aerobic treatment processes, oil and grease considerably affect oxygen mass transfer. In anaerobic processes, poor activity sludges can be developed and foam can accumulate on the surface of the water (Jeganathan et al., 2006). These factors result in biomass losses with effluent, which can significantly affect a system's efficiency (Jeganathan et al., 2006). Actions to mitigate operational problems related to high organic loads, which are commonly generated in processing industries, have been successfully developed (Bustillo-Lecompte and Mehrvar, 2015). In this context, diverse process combinations such as anaerobic, aerobic and facultative lagoons, activated sludge and

Corresponding author. E-mail addresses: [email protected] (R.G.B.d. Almeida), [email protected] (C.E.D.d. Santos), [email protected] (T.C. Lüders), [email protected] (V. Del Nery), [email protected] (C.D. Leal), [email protected] (A.D. Pereira), [email protected] (J.C. Araújo), [email protected] (R.J. Davenport), [email protected] (A.C. Barana), [email protected] (D.D. Lopes), [email protected] (M.H.R.Z. Damianovic). https://doi.org/10.1016/j.ibiod.2018.06.019 Received 13 April 2018; Received in revised form 31 May 2018; Accepted 22 June 2018 0964-8305/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. Schematic diagram of the experimental apparatus (A1: feeding entrance; A2: recirculation entrance; A3: effluent discharge; A4: recirculation exit; A5: air diffusion) (Adapted from Moura et al., 2012).

by ammonia-oxidizing bacteria (AOB). The available NO2−-N was utilized by the anammox bacteria to oxidize the remaining NH4+-N to N2, releasing NO3−-N, which will be reduced by heterotropic denitrifiers by using the residual organic matter as an electron donor (Chen et al., 2009, 2017; Wang et al., 2010). Therefore, in SNAD systems, anammox takes place as a primary process for nitrogen removal and is responsible for NO3−-N production and consequently correlates with the denitrification occurrence. Recent studies have demonstrated that COD/N ratio is an important parameter to ensure SNAD stability (Daverey et al., 2013; Chen et al., 2017). In addition, it was proved that this bioprocess is favored in attached systems, as maintaining a long sludge retention time (SRT) can develop an anammox community (Chen et al., 2009; Liang et al., 2014), as this group has a low specific growth rate (Strous et al., 1998). Nonetheless, the interruption of nitratation is not easily achieved (Yang and Yang, 2011; Yoo et al., 1999). Previous studies have concluded that other operational parameters also affect the ammonium/ nitrite oxidation rates such as pH, temperature, SRT and substrate loads (Anthonisen et al., 1976; Chung et al., 2007). When extreme changes in pH occur, the effects of substrate concentration become considerably important because of their inhibitory effects when unionized (Park and Bae, 2009). Anthonisen et al. (1976) observed that in basic conditions, unionized free ammonia (FA; NH3) concentrations of 0.1–1.0 and 10 to 150 mgFA L−1 inhibited both ammonium and nitrite oxidation, respectively. The main aim of this study was to investigate the simultaneous process of partial nitrification, anammox and denitrification for posttreatment of animal feed processing wastewater in a structured-bed reactor subjected to low aeration. In this context, this study offers some important insights into the mechanisms associated to inhibition effects on nitratation and denitrification and also the microbial characterization developed in the bioreactor, which are essential for understanding the system's performance.

trickling filters can be used (Bustillo-Lecompte and Mehrvar, 2015; Massé and Masse, 2000). However, the residual nitrogen content should be removed in post-treatment units at wastewater treatment plants (WWTP). In this context, it is essential to develop technologies which can metabolize the residual organic matter and nitrogen from this type of wastewater in order to satisfy the discharge standards. Biological treatment systems that use immobilized biomass demonstrate greater robustness to tolerate toxic and high organic loadings, minimizing biomass washout (Mijaylova-Nacheva and CanulChuil, 2006) despite the increased flow velocities (Mockaitis et al., 2014). Biomass immobilization has been successfully used to promote the simultaneous nitrification denitrification (SND) process (Moura et al., 2012; Reboleiro-Rivas et al., 2015; Santos et al., 2016; Lin et al., 2016). SND systems have gained attention due to their potential of achieving nitrogen removal rates similar to those values observed in conventional two-stage reactors. They are also cost-effective in terms of construction and operation (Moura et al., 2012; Pochana and Keller, 1999; Yoo et al., 1999). Studies showing nitrogen and organic matter removal efficiencies between 93% and 95%, respectively, were published for different types of wastewater in operating lab-scale SND reactors (Barana et al., 2013; Liu et al., 2010; Moura et al., 2012; Santos et al., 2016; Wosiack et al., 2015). In cases where there are low C/N ratios, anammox (anaerobic oxidation of ammonia) bacteria can also coexist as a complementary pathway for nitrogen removal, as observed by Barana et al. (2013) and Santos et al. (2016). Based on the fact that SND occurs due to the maintenance of a dissolved oxygen (DO) gradient in the biofilm, systems that provide a suitable DO concentration for nitrification and resistance to oxygen transfer in the biofilm interior are the most suitable (Pochana and Keller, 1999). The occurrence of the SND process by the shortened pathway has attracted considerable attention as an effective approach to reduce energy consumption (Yoo et al., 1999). This is possible by limiting the oxygen supply, aiming to minimize the nitrite (NO2-N) oxidation (nitratation) rate, while maximizing the ammonium (NH4-N) oxidation rate (nitritation) (Park and Bae, 2009). Recently, the simultaneous partial nitrification, anammox and denitrification (SNAD) process under limiting DO concentration was investigated (Chen et al., 2009). Firstly, NH4+-N was oxidized to NO2−-N 109

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bicarbonate for each mg of NH4+-N to be oxidized (Tchobanoglous et al., 2003). Phases 2 and 3 were characterized by the addition of excess alkalinity (2.5 times the recommended stoichiometric value).

2. Materials and methods 2.1. Experimental setup and inoculation The cylindrical-shaped reactor was made of acrylic. It had an internal diameter of 14.5 cm and a height of 80 cm (Fig. 1) resulting in a useful volume of 6.1 L. The diffusion of air to the liquid medium (A5) was obtained by using two porous stones connected to the Regent 8500 aquarium aerator, with two air outlets of 0.7 LO2 min−1, controlled by two needle aquarium valves to adjust the air injection into the system. Feeding and recirculation were carried out using a ProMinent Contact diaphragm pump, maximum flow rates of 19.0 L h−1 and another Gilson Miniplus peristaltic pump, with a maximum flow of 1.8 L h−1. The reactor had an internal recirculation system, with a recirculation ratio equal to 3, sufficient to ensure complete mixing. Moreover, recirculation can facilitate the mass transfer and promote the dilution of NH4+, NO2−and NO3−, resulting in a stable buffer capacity and, consequently better regulation between the combined processes. The reactor was operated under mesophilic conditions (30 °C) and filled with a structured bed consisting of 13 cylindrical structures (diameter of 3 cm and height of 60 cm) of polyurethane (PU) foam (22 g L-1 density and 92% porosity). These structures were arranged vertically inside the reactor. PU foam rods were inoculated according to (Mockaitis et al., 2012) with a mixture containing 50% (v/v) of the biomass from an up-flow anaerobic sludge bed reactor (UASB), treating wastewater from a poultry slaughterhouse and sludge from an activated sludge system of the wastewater treatment plant at the Volkswagen Motor Plant (São Carlos, Brazil). Anaerobic sludge was chosen because of its high microbial diversity (Hirasawa et al., 2008) and because it is widely used in treatment systems to remove nitrogen compounds (Souza and Foresti, 2013). However, the aerobic sludge used in this study was characterized by a significant nitrifying activity (Barana et al., 2013; Santos et al., 2016), which is important for starting systems that aim to establish SND and SNAD processes.

2.2.2. Physical-chemical analysis While monitoring the reactor, the following parameters were monitored: COD, pH, TKN (Total Kjedahl Nitrogen), NH4+-N (ammoniacal nitrogen), NO2−-N (nitrite), NO3−N (nitrate), DO and alkalinity. The alkalinity determination followed the methodology described by Ripley et al. (1986). The DO concentration was measured using the Orion 810-A+ model oximeter. NO2−-N, NO3−-N and NH4+-N analyses were performed on a Dionex ion chromatograph equipped with a conductivity detector and two different columns (IonPac® AG23 AnionExchange Column and IonPac® CG12A Cation-Exchange Column) operating at a temperature of 30 °C. To determine the anions, the flow rate was 1.0 mL min−1 and the mobile phase was the solution of sodium carbonate and sodium hydrogen carbonate (4.5 and 0.8 mM, respectively), as described by Costa et al. (2018). To determine the NH4+-N, the mobile phase consisted of sulfuric acid solution (40 mM). The other determinations followed the methods described in APHA (2012). All analytical determinations were carried out between one and three times a week, depending on the reactor's performance. 2.2.3. Operating conditions After inoculating the system, the biomass was maintained under DO concentration between 2.0 and 3.0 mgO2 L−1 for 52 days to allow the nitrifying community to develop. After detecting the NH4+-N oxidation, three operational phases (1, 2 and 3) were started, maintaining Hydraulic Retention Time (HRT) of 24 h. The DO concentration was maintained close to 1.0 mgO2 L−1 throughout the operation, aiming at maintaining nitrification and allowing the occurrence of anoxic/anaerobic processes (Yuan and Gao, 2010). At the end of the operational period, biomass samples from the reactor support material were removed and submitted to molecular microbiology analyses. The Kolmogorov-Smirnov Test (α = 0.05) was used to verify the normal distribution of total-N and COD removal efficiencies, using the OriginPro 2016®. The comparison between the data series of the three operational phases was carried out using the ANOVA One Way test and Fish Test (α = 0.05). These tools are suitable for samples with parametric distribution and of different sizes, thus determining if the data series are statistically different or not.

2.2. Monitoring the bioreactor 2.2.1. Wastewater The wastewater used for the experiment was the UASB system effluent from INCOFAP (Ibaté, Brazil), which produces animal feed from poultry slaughter residues (feather and viscera processing). According to the characterization provided by Kurian et al. (2005), animal feed production effluents are rich in total suspended solids (TSS), volatile suspended solids (VSS), chemical oxygen demand (total and soluble COD), ammoniacal nitrogen, phosphate, oil and grease. These compounds are related to the presence of carbohydrates, proteins, fats and amino acids from the residual blood, as well as feather and viscera processing. The reactor was operated in three operational phases, defined by the quality of the wastewater from different collections. In Phase 1, stoichiometric alkali was added: 7.14 mg of CaCO3 in the form of sodium

2.2.4. Analysis of microbial community In order to investigate the presence of nitrifiers, anammox bacteria and denitrifiers, DNA was extracted from the biomass sample (10 mL taken from the reactor at the end of Phase 3) using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, USA) according to the manufacturer's instructions. Concentration and purity of the DNA extract was determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific). Polymerase Chain Reaction (PCR) reactions were performed using the following primers (Table 1). After the PCR, the presence and

Table 1 Primers used to detect AOB, NOB, anammox bacteria and denitrifiers. Primers

Target gene

Specificity

Annealing temperature (°C)

Fragment size (bp)

Reference

Pla46F/Amx820R amoA-1F amoA-2R Nitro-1998F Nitro-1423R NTPSA F NTPSA R nosZF nosZ1622R nirS cd3AF nirS R3cd

16S rRNA Ammonia mono oxigenase

Anammox, “Ca. Brocadia” “Ca. Kuenenia” Ammonia oxidizing bacteria

56 57

774 491

Schmidt et al. (2003) Rotthauwe et al. (1997)

16S rRNA

Nitrobacter sp

58

367

Graham et al. (2007)

16S rRNA

Nitrospira sp

60

151

Kindaichi et al. (2006)

Nitrous oxide reductase

Denitrifiers

55

494

Enwall et al. (2005)

Nitrite reductase

Denitrifiers

50

406

Throbäck et al. (2004)

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maintenance of effluent pH values above 8.0 (Fig. 2b) was associated to the reduction in the denitrifying activity (Fig. 2c) and NO2−-N accumulation (Table 2). Park and Bae (2009) tested the influence of FA, free nitrous acid (FNA) and the limitation of DO concentration on the NO2− accumulation observed in partial nitrification systems. The authors observed that the NO2− accumulation is more evident when FA accumulation is associated with the limitation in the DO. These operating conditions were also established in the present study (Fig. 2c and d), corroborating with the hypothesis raised of the inhibitory effect of NO2−-N under heterotrophic denitrification. The increase in pH interferes with the distribution of the NH4+-N form in solution, favoring the FA formation, which at high concentrations can be pointed out as the main interference to the nitrification process (Anthonisen et al., 1976). Bae et al. (2002) and Liu et al. (2010) investigated the FA inhibition over NOB community in a nitrifying reactor, considering the batch and continuous operation mode, respectively. These authors observed inhibition on Nitrobacter genera when the FA concentration varied from 0.1 to 4 mgFA L−1. ANOVA statistical test results enabled us to identify that the only data set that can be considered as different was the total-N removal observed for Phase 1, corroborating with the hypothesis of inhibition of the denitrification process due to an increase in pH associated with excess alkalinity. Thus, from the point of view of total-N removal, Phase 1 can be considered as the one with the best performance. Differently from that observed for the total-N removal, the COD removal remaining in the UASB effluent was more homogeneous for Phase 1 with a coefficient of variation equal to 32%. Phase 1 was also characterized by the higher COD removal efficiency with an average of 63% and a maximum of 92%. Phases 2 and 3 presented COD removal efficiencies of 38 ± 27% and 48 ± 31%, respectively. The low organic matter removal efficiency in the reactor is related to the low organic matter removal potential of the organic content of the anaerobic effluent (BOD/COD ratio close to 0.55), which can be seen by the residual COD observed in the effluent of the post-treatment unit. Philips et al. (2002) proposed that the stoichiometric ratio between COD/ NO3−-N in the heterotrophic denitrification process should be equal to or greater than 4. Thus, Figure A1 (Supplementary Material) presents the comparison between the COD removed in the system and the COD calculated stoichiometrically (COD/NO3−-N = 4.5) for the cases in which all nitrogen removal was from heterotrophic denitrification via NO3−-N or NO2−-N. It is worth mentioning that heterotrophic denitrification via NO2−-N allows a 40% reduction of COD required when compared to NO3−-N denitrification (Tchobanoglous et al., 2003). Therefore, it can be observed that for all operational conditions the required COD had a significantly higher value than COD removed from the system, indicating the development of other processes of total-N removal, not related to heterotrophic denitrification. Thus, a mass balance of the nitrogen content metabolized in the oxidation processes of ammonia, heterotrophic denitrification and anammox was performed for each experimental phase (Table 3). The anammox process became the main pathway of nitrogen removal in Phases 2 and 3 and was responsible for more than 50% of the total N-removed in the studied system (Table 3). This fact is probably due to the toxic effect of NO2− under the denitrification process, which resulted in a drop in the Total-N loading denitrified (Table 3). Furthermore, a visual alteration of biomass coloration was observed in the reactor by comparing the beginning of the operation to the end of the operation, as presented in Figure A2 (Supplementary Material). As NO3− reduction by the heterotrophic denitrification pathway occurred until the end of the reactor's operation, despite the low availability of COD (Table 3), it can thus be suggested that biomass could be used as an endogenous carbon source for heterotrophic denitrification. This biomass loss, markedly observed in Phase 3 may also have potentiated the toxic effect of nitrite on the denitrifying community, as discussed by Beccari et al. (1983). These authors observed that in NO2−-N concentration varying from 20 to 25 mg L−1 and a pH range of 7–8,

size of amplification products were determined by agarose (1%) gel electrophoresis of 5 μL aliquots of the PCR products. A portion of DNA extracted from the sludge sample at the end of phase 3 was used for PCR amplification (with primers 515F and 926R, targeting the V4 and most of the V5 region of the 16S rRNA gene of Archaea and Bacteria), library construction and sequencing using the Ion Torrent platform. The sequencing in the Ion Torrent PGM (400bp) was performed at the School of Engineering & Geosciences (Newcastle University) using the 316™ ion chip following the manufacturer's instructions (Life Technologies, USA). Raw sequences were analysed using a QIIME (v 1.7.0) bioinformatics pipeline. After the quality filter (minimum quality score of 20, perfect match to sequence barcode and primer), remaining sequences were clustered in the Operational Taxonomic Unit (OTU) at 97% similarity level, and the representative sequences were taxonomically assigned using the SILVA database (Quast et al., 2013). The final results were given in relative abundance (%). 3. Results and discussion 3.1. Performance of the reactor Table 2 shows the characteristics of the reactor influent and effluent during the operational phases. The reactor operation lasted 236 days, having had a previous period of adaptation of nitrifying biomass (52 days). The factors that differentiated the three operational conditions were applied organic and nitrogen loads and also different values of influent alkalinity (Table 2). In all phases, total-N removal occurred (Fig. 2a), however the process was unstable. For example, the minimum coefficient of variation was 46% for Phase 3, which was characterized by greater heterogeneity of the data. The UASB reactor effluent, collected during the operational period, presented a variable composition, causing changes in the quality of the influent and consequently in the reactor performance. Phase 1 showed the highest average efficiency of total-N removal (48 ± 24%), reaching a maximum value of 79%, under a C/N ratio of 0.28. Moura et al. (2012) and Santos et al. (2016), who operated similar systems treating sanitary sewage and Barana et al. (2013) and Wosiack et al. (2015), who operated systems and wastewater from similar origins, obtained average total-N efficiencies above 60%, under C/N ratios varying from 2.5 to 11.6, favoring the establishment of heterotrophic denitrification. Although the addition of excess alkalinity allowed higher stability of the system for total-N removal in Phases 2 and 3 (Fig. 2a), the Table 2 Characterization of the reactor influent and effluent for the three operational phases. Parameter

Phase 1

Phase 2

Phase 3

Duration (days)

62

75

47

INFLUENT Total COD (mg L−1) Soluble COD (mg L−1) NH4+-N (mgL−1) TKN (mgL−1) Alkalinity (mgCaCO3 L−1) CODsoluble ratio/N

219 ± 103 89 ± 40 231 ± 40 315 ± 53 1545 ± 337 0,28

159 ± 72 102 ± 27 192 ± 55 255 ± 67 3404 ± 267 0,41

43 ± 26 38 ± 20 124 ± 26 146 ± 18 1250 ± 113 0,26

EFFLUENT Total COD (mg L−1) Soluble COD (mg L−1) NH4+-N (mgL−1) TKN (mgL−1) NO2−-N (mgL−1) NO3−-N (mgL−1) Alkalinity (mgCaCO3 L−1)

33 ± 15 30 ± 15 48 ± 31 65 ± 49 37 ± 37 84 ± 56 256 ± 167

78 ± 21 61 ± 25 52 ± 42 76 ± 58 67 ± 31 46 ± 48 256 ± 167

17 ± 15 23 ± 23 39 ± 4 45 ± 10 56 ± 8 17 ± 20 862 ± 130

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Fig. 2. (a) Box-Plot graphs for ammonia oxidation efficiency (blue), denitrification efficiency (red) and total-N removal (black) at all phases of the operation. (b) Box plot graphs for influent pH (black) and effluent pH (blue) at all phases of the operation. (c) Box-Plot graphs for effluent concentrations of nitrate (black) and nitrite (blue) at all phases of the operation. (d) Box plot graphs showing free ammonia effluent concentration in all phases of the operation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Table 3 Nitrogen mass balance evaluation of participation of different processes. Variables

Phase 1

Phase 2

Phase 3

Nitrogen loading rate (kgN m−3 day−1) Total-N loading nitrified (kgN m−3 day−1) Total-N loading denitrified (kgN m−3 day−1) Total-N loading removed by anammox (kgN m−3 day−1) Total-N loading in effluent (kgN m−3 day−1)

0.307 0.149 0.087 0.062

0.249 0.086 0.034 0.042

0.149 0.041 0.014 0.028

0.158

0.173

0.107

Table 4 PCR amplification results with different primer pairs to detect AOB, NOB, anammox bacteria and denitrifiers (for the target genes of each pair of primers see Table 1). Pair of primers

Specificity

Sludge sample after phase 3

amoA-1F/amoA-2R Pla46F/Amx820R

Ammonia oxidizing bacteria Anammox, “Ca. Brocadia” “Ca. Kuenenia” Nitrospira sp. Nitrobacter sp

+

denitrifiers denitrifiers

+ +

NTPSA F/NTPSA R Nitro-1998F/Nitro1423R nosZF/nosZ1622R nirS cd3AF/nirS R3cd

Nitrogen mass balance obtained considering the anammox stoichiometry: 1.32 NO2− consumed/1 NH4+ oxidized, and 0.26 NO3− formed.

complete denitrification was obtained with a biomass content varying from 500 to 1000 mgVSS L−1. Nevertheless, for the same conditions, but considering a biomass concentration varying from 100 to 150 mgVSS L−1, remarkable inhibition of denitrification was attained.

+

(+) Positive result, amplification product was visualized on agarose gel. (−) Negative result, no amplification product was formed.

present (Fig. 4). The main identified microrganisms in the reactor at the end of operation are shown in Figures 4 and 5. The Ion Torrent sequencing results revealed that AOB, anammox, denitrifiers, among others (fermenting bacteria, methanogenic archaea, etc.) were present in the reactor after the tested conditions (Figs. 3 and 4). Among the sequences related to bacterial groups potentially involved in the nitrogen cycle, AOB were detected in an abundance from 0.4 to 0.6%, expressed by sequences related to Nitrosomonadales and Nitrosomonas (Figs. 3 and 4, respectively). This abundance appears to be significant by considering the existence of a mixotrophic biofilm. Persson et al. (2017) observed AOB abundance of 0.3–0.4% in a moving bed biofilm reactor (MBBR) operating in partial nitrification/anammox conditions. Regarding NOB, sequences related to Nitrobacter sp or Nitrospira sp. were not detected by

3.2. The main identified microbes in the reactor as shown by PCR and ion torrent sequencing The PCR results using specific primers to detect AOB, NOB, anammox bacteria and denitrifiers (using primers for nitrite reductase and nitrous oxide reductase genes) are shown in Table 4. Positive results were obtained from the amplification with primers for anammox bacteria, Nitrobacter and denitrifiers confirming the presence of these groups in the reactor's biomass. Negative results were obtained with the amoA primers suggesting that AOB were not present in the biofilm at the end of the operation (Phase 3), or could be present but below the PCR detection limit. In fact, Ion Torrent sequencing results retrieved sequences related to Nitrosomonadaceae, indicating that AOB were 112

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Fig. 3. Taxonomic composition of the microbial communities at the order level. Biomass was sampled from the reactor at the end of operational Phase 3.

metabolism (Hug et al., 2013) were detected in high abundance (from 8.0 to 10.0%). Members of the phyla Bacteroidetes (Delbés et al., 2000) and Chloroflexi (Ariesyady et al., 2007) are known as hydrolytic and fermentative bacteria and have been detected in anaerobic reactors treating sewage (Li and Lu, 2017). Within this phylum, sequences related to Anaerolineales were identified (Fig. 4). This order comprises anaerobic and heterotrophic bacteria (involved in saccharide degradation) reported in different environments including anaerobic reactors (Yamada et al., 2006) and anammox enriched reactors (Pereira et al., 2017). Sequences related to Firmicutes (order Clostridiales) accounted for 4.0% of the total sequences (Fig. 4). Clostridiales affiliated with the class Clostridia has a diverse metabolism that can ferment polysaccharide to produce VFAs for methanogenesis (Vos et al., 2009; Sundberg et al., 2013). Methanobacterium, which are hydrogenotrophic methanogenic archaea (MA), were also identified (accounting for 2.5% of the total) (Figure 5). Sulfate-reducing bacteria (SRB) with sequences related to Syntrophobacterales were identified (2.0% of the total) (Fig. 4). Members of Syntrophobacterales, such as Desulfovibrio, Desulfomicrobium and species of Syntrophobacter and Smithella, are acetogenic bacteria that can degrade propionate (Muyzer and Stams, 2008) and produce hydrogen (H2) in syntrophic association with MA (Muyzer and Stams, 2008). The presence of methanogens and SRB can be explained by the fact that 50% of the inoculum used in this study came from a UASB reactor. Moreover, during the whole operational period,

sequencing analysis. Nonetheless, positive PCR results with primers targeting Nitrobacter 16SrRNA were observed (Table 4), suggesting that NOB were present in the structured-bed reactor. Since primers applied in each technique were different, targeting different regions of 16SrRNA gene, this might be the reason for the opposite findings observed. Denitrifying bacteria was the most abundant group in the present study (Figs. 3 and 4). Sequences related to the orders Burkholderiales, Rhodocyclales, Xanthomonadales and Pseudomonadales, which are heterotrophic denitrifiers (Heylen et al., 2006; Satyanarayana et al., 2012), were detected at a high frequency (accounting for 22.1%). These groups have been detected in an anammox reactor treating anaerobically pretreated municipal wastewater (Fernandes et al., 2018). Anammox bacteria were also detected in the present study, confirming the results obtained from the PCR analysis (Table 3). Sequences belonging to the Planctomycetes phylum, related to Candidatus Anammoximicrobium, were identified and accounted for 2.0%. This genus was firstly described by Khramenkov et al. (2013). Regarding the other metabolisms, sequences related to Sphingobacteriales and Flavobacteriales, within the phylum Bacteroidetes, were detected and identified accounting for 18.5% and 4.1%, respectively. Bacteroidetes is composed of bacteria that are involved in the hydrolysis and acidogenesis steps of anaerobic digestion. Members of the phylum Chloroflexi, which includes bacteria with diversified

Fig. 4. Microbial composition at the genus level. The results are presented as the percentage of sequences assigned to each genus among the total sequences in a sample. 113

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References

the structured-bed reactor was fed with effluent from the UASB system applied to the animal feed processing wastewater treatment. This fact, associated with the anoxic/anaerobic conditions established in the inner biofilm layers led to the development of these microbial groups. Specifically concerning SRB activity, the wastewater used in this study was pre-treated in a UASB reactor. Therefore, sulfide was formed during this step (data not shown), which could be oxidized to sulfate due to continuous aeration in the structured-bed reactor. The formed sulfate could then be reduced in the anaerobic portion of the biofilm.

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4. Conclusions In pH values above 8.0, FA concentration in the liquid medium increased, which together with the reduced availability of DO, caused NO2− accumulation in the reactor, as a function of NOB inhibition. The NO2−-N accumulation, with concentrations exceeding 100 mgN L−1, was responsible for the toxicity to the denitrifying biomass. The anammox activity, developed according to the characteristics of the wastewater and the operating conditions established in the reactor, was responsible for Total-N removal efficiencies of 42%, 55% and 67% of the nitrogen loads applied in Phases 1, 2 and 3, respectively. Sequencing analysis revealed that the microbial community present in the system at the end of the operational period was complex and diverse, with heterotrophic denitrifiers, AOB and anammox bacteria coexisting with hydrolytic/fermenting, SRB and methanogenic archaea. These results associated with operational monitoring data reveal the potentiality of the structured-bed reactor subjected to low aeration and recirculation to perform the SNAD process and indicates its suitable application as a post-treatment technology. Conflicts of interest statement The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. Author names: Ricardo Gabriel Bandeira de Almeida; Carla Eloísa Diniz dos Santos; Taíssa Colucio Lüders, Valéria Del Nery; Cintia Dutra Leal, Alyne Duarte Pereira, Juliana Calábria Araújo; Russel J. Davenport, Ana Cláudia Barana; Deize Dias Lopes; Márcia Helena Rissato Zamariolli Damianovic. 5. Declarations of interest None. Acknowledgements This study was supported by the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (grant number 2015/ 06246-7). We would also like to thank INCOFAP pet food industry (Ibaté, São Paulo, Brazil), which provided the wastewater used in this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.ibiod.2018.06.019. 114

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