Recirculation and Aeration Effects on Deammonification Activity
Angélica Chini, Airton Kunz, Aline Viancelli, Lucas Antunes Scussiato, Jéssica Rosa Dias & Ismael Chimanko Jacinto Water, Air, & Soil Pollution An International Journal of Environmental Pollution ISSN 0049-6979 Volume 227 Number 2 Water Air Soil Pollut (2016) 227:1-10 DOI 10.1007/s11270-016-2765-7
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Author's personal copy Water Air Soil Pollut (2016) 227: 67 DOI 10.1007/s11270-016-2765-7
Recirculation and Aeration Effects on Deammonification Activity Angélica Chini & Airton Kunz & Aline Viancelli & Lucas Antunes Scussiato & Jéssica Rosa Dias & Ismael Chimanko Jacinto
Received: 16 October 2015 / Accepted: 18 January 2016 / Published online: 1 February 2016 # Springer International Publishing Switzerland 2016
Abstract Deammonification process has been studied as an alternative technology for nitrogen removal. This process consists of the association between nitrifying and anammox bacteria, in which the process success is related to aeration, recirculation, and reactor configuration. Considering this, the present study aimed to evaluate the performance of an expanded granular sludge bed (EGSB) reactor on nitrogen removal by deammonification process. Established in a single reactor, it considered the effects of recirculation rate and variation of dissolved oxygen (DO) concentration in microbial community and nitrogen removal efficiency. Thus, two independent tests were conducted: (T1) high recirculation flow rate, performed at 43 L d−1 (Qr/ Qin = 16), aeration of 30 mLair min−1 L−1reactor, and conducted during 16 days; (T2) low recirculation flow rate performed at 6.7 L d−1 (Qr/Qin = 2.5), operated for 55 days, divided into three aeration phases: (T2a) 30 mLair min−1 L−1 reactor, (T2b) 20 mLair min−1 L−1 reac−1 −1 L reactor. Results showed tor, and (T2c) 30 mLair min that in T1 the high recirculation rate favored nitrifying bacteria prevalence, intensified by reactor turbulence A. Chini (*) : A. Kunz : L. A. Scussiato Western Paraná State University, Rua Universitária, 2069, JD. Universitário, Prédio de Desenvolvimento de Protótipos, Cep: 85819-110 Cascavel, PR, Brazil e-mail:
[email protected] A. Kunz Embrapa Swine and Poultry, Concórdia, SC, Brazil A. Viancelli : J. R. Dias : I. C. Jacinto Contestado University, Concórdia, SC, Brazil
and anammox granules disintegration, changing activity from deammonification to a nitrification process. In addition, T1 reached up to 350 ± 100 mgN L−1 d−1 nitrogen removal rate (NRR). For T2, at low recirculation rate, deammonification process was successfully established with a NRR of 490 mgN L−1 d−1 at Qr/ Qin = 2.5 and air flow rate of 20 mLair min−1 L−1reactor. Keywords Anammox . Deammonification . EGSB reactor . Nitrification . Nitrogen removal
1 Introduction Nitrogen compounds present in effluents (e.g., animal manure) must be under concern because of the potential of environmental pollution and health problems, such as water bodies eutrophication and human methemoglobinemia disease (Akpor and Muchie 2011). Conventional technologies for nitrogen removal from wastewater, using nitrification and denitrification processes are expensive, especially for aerobic treatment that consumes a high amount of oxygen and can generate a high sludge volume (Bagchi et al. 2012). For this reason, alternative treatment strategies have been studied, and one of the promising technologies is the deammonification process. This process consists of the combination of partial nitritation (PN) and anaerobic ammonium oxidation (anammox) processes (Xu et al. 2015), either in separate or single reactors. According to Li et al. (2015), this
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process consumes 63 % less oxygen when compared to conventional nitrification–denitrification process. The success of this technology is related to the cooperation of two groups of microorganisms: partial nitrifiers (aerobic) and anammox (anaerobic). Ammonia oxidation by PN (Eq. 1) generates substrate (ammonia and nitrite) to anammox activity (Eq. 2). The overall stoichiometry of deammonification process is shown in Eq. 3 (Daverey et al. 2013; Buha et al. 2015). PN :
þ − 2N H þ 4 þ 1:5O2 → N H 4 þ N O2 − H 2 O
þ 2H þ
ð1Þ
− Anammox : N H þ 4 þ 1:3N O2 → 1:02N 2
þ 0:26N O−3 þ 2 H 2 O
ð2Þ
Deammonification : N H þ 4 þ 0:85O2 → 0:44N 2 þ 0:11N O−3 þ 1:43 H 2 O þ 1:14 H þ
ð3Þ
Several operational parameters may influence PN, as temperature, pH, free ammonia and DO (De Prá et al. 2012). One special parameter to be considered during the reactor operation is the DO concentration, because DO influences both nitritation and anammox processes. Wei et al. (2014) affirms that for a stable PN process, the DO concentration has to be between 0.5 and 1.0 mgO2 L−1. Ruiz et al. (2003) concludes that with a DO concentration of 1.7 mgO2 L−1, there is accumulation of nitrite, which can inhibit the anammox process. DO bellow 1.5 mgO2 L−1 is favorable for PN, reducing the partial nitrification rates and generating a substrate nearby anammox stoichiometry (Zhu et al. 2008; De Prá et al. 2012). Another important factor on deammonification success is the recirculation rate. High recirculation can improve oxygen mass transference in the reactor, but it may also wash out sludge granules, with possible operational failure and reduction of sludge activities (Liang et al. 2015; Pereboom 1997). Consequently, the liquid up flow velocity cannot be compromised, because this velocity must be sufficient for mixing the sludge. The anammox granules shearing can occur, increasing solid concentration in the effluent and also nitrite
accumulation in the reactor, thus not contributing to the stability of the anammox process (Arrojo et al. 2008). Tang et al. (2013) developed a mathematical models based on compression and expansion of the granule in an up flow reactor to estimate the nitrogen conversion rate of bacteria with anammox activity. They showed that the high removal rates occur in packing density of 50–55 %. The reactor configuration also influences the process. Suspended bed reactors have been used for manure treatment (Cui 2012). However, this reactor type shows some problems as resistance to mass transfer, which promotes the occurrence of dead zones. Considering this, the expanded granular sludge bed reactor (EGSB) can be used to overcome such configuration problems. The EGSB improves mass transfer between substrates in wastewater and granular microorganisms sludge, enhancing the treatment efficiency. Therefore, with high recirculation rates and an elevated height/ diameter ratio, it is possible to achieve high superficial velocity (Jeison and Chamy 1999; Zheng et al. 2013). Furthermore, the EGSB reactor has the advantages of allowing working with high recirculation rates, restricted air flow rates and it also promotes more contact between substrate and biomass (Ren et al. 2014; Yang et al. 2015). Additionally, this configuration avoids biomass loss, which is crucial to the process stability (Kato et al. 2003). Considering this, the present study aims to evaluate the performance of an EGSB reactor on nitrogen removal by deammonification process established in a single reactor. The effects of liquid recirculation rate and DO concentration on the microbial community and nitrogen removal efficiency are also considered.
2 Material and Methods 2.1 Experimental Design A schematic representation of the experimental reactor, operated in laboratory scale, is presented at Fig. 1. A cylindrical reactor was used with internal diameter of 5 cm and 60 cm height, 1 L of working volume and containing plastic biofilm carriers (Kaldnes, 55 g w.v−1), operated as an EGSB reactor. Aeration was provided using an air pump (A230, Big Air) connected to an air
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EPDM diffuser and measured by an air flowmeter (Gilmont, GF-9260). The reactor was operated at hydraulic retention time (HRT) of 9 h, with intermittent aeration cycles (15 min on /15 min off) controlled by programmable logical controller (PLC Dexter, model lDX series 100) at 25 ± 1 °C and nitrogen loading rate (NLR) of 800 ± 20 mgN L−1 d−1. An Imhoff cone (1 L) was used as a settler after the reactor, aiming to retain the microbial biomass carried out with the effluent and the settled biomass recirculated back to the reactor. The recirculation flow rate (Qr) was adjusted as required. For liquid circulation and recirculation, peristaltic pumps (Milan BP-200 and BP-600) were used. The reactor was fed with synthetic wastewater, containing (mg L−1) (NH4)2SO4 (1416), KH2PO4 (100), NaHCO3 (2912), Na2CO3 (391), MgSO4 7H2O (60), FeSO4 · 7H2O (8), CaCl2.2H2O (8). Trace nutrient solution was added by 100 μL L−1, according to Magrí et al. (2012).
2.2 Inoculum Characterization Inoculum containing bacteria with anammox activity and bacteria with nitrifying activity were obtained from lab-scale reactors, which had been operated for over a year (De Prá et al. 2012; Casagrande et al. 2013). The sludge ratio used for reactor inoculation between anammox and nitrifying biomass was determined by their specific nitrogen consumption rates obtained in batch tests of activity. Anammox and nitrifying activities (Table 1) were tested in batch tests (3 h), with internal liquid recycle (3 mL min−1), 200 mL of biomass were used for anammox batches (Puyol et al. 2013). For nitrifying batches, air was injected at 30 mLair min−1Lreactor−1 and 80 mL of biomass were used (Li et al. 2014a). Samples were collected at t0 and after every 30 min. For each collected sample, pH, DO and temperature were immediately determined. After it, they were sent to NH3–N, NO3−–N, NO2−–N and alkalinity analysis. At the end of the batch tests, volatile suspended solids (VSS) were determined. All samples were analyzed according to APHA (2012). Nitrite and ammonia consumption rate (r) and specific nitrite and ammonia consumption rate (μs) were determined from substrate concentration versus time linear regression obtained in batch tests (Li et al. 2014a, b; Puyol et al. 2013).
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The inoculum characterization is described at Table 1. Nitrifying and anammox biomasses were placed simultaneously inside the reactor for deammonification experiments. 2.3 Recirculation and DO Influence Tests Two tests were conducted to evaluate the influence of r e c i r c ul at i on a nd D O c on c en t r a t i o n on t h e deammonification process. Each test was conducted in a separate experiment, with independent inoculation practices. Test 1 (T1) had a recirculation flow rate of 43 L d−1 (Qr/Qin = 16, this ratio was established for the reactor to behave as a traditional EGSB), aeration of 30 mLair min−1 L−1 reactor and it was conducted during 16 days. Test 2 (T2) had a recirculation flow rate of 6.7 L d−1 (Qr/Qin = 2.5), was operated for 55 days, divided into three phases: (T2a) (1–11 days) aeration at 30 mLair min−1 L−1 reactor (DO 0.80 ± 0.30 mgO2 L−1), (T2b) (12–35 days) with aeration at 20 mLair min−1 L−1 −1 reactor (DO 0.56 ± 0.13 mgO 2 L ), and (T2c) (36– 55 days) with aeration at 30 mLair min−1 L−1 reactor (DO 0.69 ± 0.16 mgO2 L−1). 2.4 Analytical Methods Influent and effluent samples were daily collected and analyzed for total ammoniacal nitrogen (TAN expressed as NH3–N), NO3−–N, NO2−–N and alkalinity according to APHA (2012). DO (YSI 55) and pH (Hanna, pH 21) were monitored in aerobic and anoxic phases. Nitrifying and anammox bacteria quantification were performed by quantitative polymerase chain reaction (qPCR), using SYBR green and primers as described by Braker et al. (1998) and Schmid et al. (2008), respectively. qPCR reactions were performed using delta delta quantification cycle (ΔΔCq) comparation.
3 Results and Discussion 3.1 Start-up and Recirculation Influence (T1) As the inoculums used in the experiment came from a nitrifier reactor and an anammox reactor with established activities. The EGSB reactor start-up took
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Fig. 1 Schematic representation of EGSB reactor used in the present study operated in laboratory scale
reaching 350 ± 100 mgN L−1 d−1, achieving total nitrogen (TN) removal efficiency of 42 ± 16 % (Fig. 2). However, after the 8th operational day, the reactor presented a decrease in NRR and an increase in NO2−–N accumulation, showing a prevalence of nitrifying activity instead of deammonification, which can be attested by the increase of O2 stoichiometric coefficient (Fig. 3) (Daverey et al. 2013). Batch tests also showed that
only 3 days to increase NRR, reaching stability on effluent NH3–N concentration (Fig. 2) and stoichiometric coefficients (Fig. 3) after the 4th operation day. The decreasing in NH3–N effluent concentration and also NO2−−N and NO3−–N effluent low concentration (Fig. 2) indicated that the deammonification process was prevalent in the reactor until the 6th day. At this period, the nitrogen removal rate (NRR) increased until
Table 1 Nitrifying and anammox biomasses characterization before reactor inoculation for T1 and T2 T1
T2
Parameters
Unit
Nitrifying biomass
Anammox biomass
Nitrifying biomass
Anammox biomass
Ammonia consumption rate
mgN L−1 h−1
114.1
19.5
100.4
19.5
Specific ammonia consumption rate (μNH3-N) Nitrite consumption rate
mgNH3–N gSSV−1 h−1
107.17
20.11
102.4
19.38
mgN L−1 h−1
-
30.0
-
28.6
Specific nitrite consumption rate (μNO2−-N) Bacteria quantification
mg NO2−– N gSSV−1 h−1 gc L−1a
-
31.03
-
14.22
1.05 × 108
4.65 × 109
1.22 × 105
5.55 × 104
a
genome copies L−1
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Fig. 2 a Influent and effluent nitrogen species concentration; nitrogen loading (NLR) and removal (NRR) rates during operation time during test 1. b Dissolved oxygen (DO) during operation time at T1
ammonium and nitrite consume rate decreased (Table 2). The predominance of nitrification process is also supported by bacteria quantification results. They showed that nitrifying bacteria increased around 4
log10 from the 5th to 16th day, while the anammox bacteria community decreased around 3 log10 during the same period (Table 2). Nitrifying bacteria growth was probably favored by the recirculation ratio (Qr/Qin = 16) of the effluent when
Fig. 3 Stoichiometric coefficients during operation time in test 1 named study compared with Daverey et al. (2
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Time
Inoculum
rNH3–N (mgNH3–N L−1 h−1) μNH3–N (mgNH3–N gSSV−1 h−1) rNO2−–N (mg NO2−–N L−1 h−1) μNO2−–N
NO2−–N −1
−1
−1
Day 5
Day 16
19.45
19.53
8.60
20.11
–
–
30.02
28.66
10.58
31.03
–
–
Nitrifying (gc L )
1.05 × 108
7.10 × 106
2.06 × 1010
Anammox (gc L−1)
4.65 × 109
3.90 × 1010
7.65 × 107
(mg
gSSV
h )
back into the reactor, which intensified the turbulence inside the reactor and consequently could favor granules disintegration (Tatari et al. 2013; Xiong et al. 2013). Figure 4 shows the flocs characteristics at 1st and 16th days, respectively. At the beginning of experiments, flocs were granular; however, at the 16th day, they were fluffy, a characteristic form of nitrifying bacteria predominance (López-Palau et al. 2011). Li et al. (2014a, b) showed that decreasing the granule size also decreases the reactor efficiency. This was also observed in the present study, where on the first day the removal rate was 500 mg N L−1 d−1 while on the 16th day it was 200 mgN L−1 d−1. Besides, the recirculation could have contributed to a better oxygen mass transference to nitrifying bacteria, increasing its activity and growth (Wei et al. 2014). In this way, the reactor was discontinued and a new reactor was inoculated using a low recirculation ratio.
(Table 3), which was 7.19 and 13.11 mgN L−1 h−1 lower than inoculum. NRR average was 260 ± 50 mgN L−1 d−1 (Fig. 5), with a nitrogen removal efficiency of 35.80 ± 9.60 %. Based on these results, it is possible to conclude that anammox activity decreased inside the deammonification reactor. The anammox process was probably inhibited whereas nitrifying bacteria were favored by the DO concentration (0.87 mgO2 L−1), changing the nitrogen compounds ratio in the reactor. The bacteria quantification results also corroborated this statement, showing that nitrifying bacteria community increased 1 log10 and anammox bacteria decreased 1 log10 after 10 operational days (Table 3). These results indicate that nitrification process was predominant in the reactor at T2a phase.
3.2 Start-up at low Recirculation Ratio and Aeration Influence (T2)
In phase b, the reactor aeration flow rate was decreased from 30 to 20 mLair min−1 L−1 reactor. At this phase the average amount of ammonium, nitrite, and nitrate in the effluent were 77.86 ± 5.34 mg L−1, 19.15 ± 6.09 mg L−1 and 14.08 ± 3.74 mg L−1, respectively (Fig. 5). Comparing to the ammonium concentration in the effluent during phase T2a (151.21 ± 26.37 mg L−1) and T2b, the results indicate that in phase T2b the removal percentage was 94.2 % higher than in phase T2a. The average NRR was 490 ± 30 mgN L−1 d−1, with a nitrogen removal efficiency of 62.20 ± 2.71 % (Fig. 6). Stoichiometric coefficients were close to those described in literature (Daverey et al. 2013), demonstrating the occurrence of deammonification process in EGSB reactor (Fig. 6). In addition to these results showing the reactor improvement on nitrogen removal efficiency, the anammox consume rates of ammonium and nitrite (determined at 21st day) showed higher values than
The reactor was reinoculated and operated for 55 days at low recirculation ratio (Qr/Qin = 2.5) to avoid floc disintegration. DO was also studied to evaluate the influence in the activity and microbial community at three different stages. 3.2.1 Phase T2a T2a consisted of 11 days of operation. During this period, ammonia concentration in the effluent decreased from 195.11 to 114.70 mg L−1 (Fig. 5). However, nitrite and nitrate concentration increased from 7.80 to 32.50 mg L−1 and from 1.25 to 47.5 mg L−1, respectively. In addition, at 10th day the anammox activity presented an ammonium nitrite consume rates of 12.34 mgN L−1 h−1 and 15.55 mgN L−1 h−1 respectively
3.2.2 Phase T2b
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Fig. 4 Flocs details in the EGSB reactor at the test 1. a Granules structure at first day of operation. b Fluffy flocs at the 16th operation day
those obtained at T2a (Table 3), indicating a better reactor performance. Besides, the anammox bacteria community increased 2 log10 along the 21 operational days (Table 3). Meanwhile, nitrifying bacteria community decreased 1 log10. For the deammonification process in this phase, it was necessary 0.88 mol of O 2 per mol of NH 3
removed, reducing 56 % of the oxygen required for ammonia removal if compared to a traditional nitrification/denitrification process, which needs 2 mol of O2 per mol of NH3 removed (Van der star et al. 2011). Recent studies have shown that the deammonification process could have 65 % of reduction in aeration energy when compared with the
Fig. 5 a Influent and effluent nitrogen concentrations; nitrogen loading rate (NLR) and removal rate (NRR) during operation time in T2. b Dissolved oxygen (DO) during operation time at T2
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Table 3 Anammox activity batch tests, ammonium and nitrite consume velocities, and nitrifying and anammox bacteria quantification by qPCR, during operation time in T2 Time
Inoculum
Day 10
Day 21
Day 51
rNH3–N (mg NH3–N L−1 h−1)
19.53
12.34
18.59
9.24
μNH3–N (mg NH3–N gSSV−1 h−1)
19.38
–
–
–
rNO2−–N (mg NO2−–N L−1 h−1) μNO2−–N (mg NO2−–N gSSV−1 −1
28.66
15.55
24.92
10.90
14.22
–
–
–
Nitrifying (gc L )
1 22 × 105
5.20 × 106
6.34 × 105
1.10 × 106
−1
4
3
5
3.67 × 105
Anammox (gc L )
h−1)
5.55 × 10
classical process (nitrification–denitrification) (Van Der Star et al. 2011; Gude 2015. In addition, this phase presented stability in the reactor and proved that the deammonification process was well established with stoichiometric coefficients according to what is presented in literature (Fig. 6). 3.2.3 Phase T2c In order to check if deammonification process could keep the activity and reduce the residual ammonia concentration from the reactor effluent in phase T2b, air flow rate was increased again from 20 to 30 mLair min−1 L−1 reactor. Thus, on the first days of phase c (37–41 operational days) ammonium effluent concentration decreased from 112.38 to 48.30 mg L−1. However, on the 42nd operational day, achieving concentrations of 133.34 mg NH3–
4.25 × 10
1.19 × 10
N L−1 (Fig. 5) started to accumulate in the reactor. This ammonia accumulation indicates that the amount of ammonia which oxidized to nitrite was above the amount taken by anammox bacteria that are inhibited by nitrite (40–350 mg NO2 −–N L −1 ) (Fux 2003; Dapena-mora et al. 2007). Furthermore, nitrite and nitrate concentration in the effluent ranged from 16.57 to 73.55 mg L−1 and from 12.77 to 28.55 mg L−1 (Fig. 5), respectively. The average NRR was 330 ± 90 mgN L−1 d−1, with a nitrogen removal efficiency of 41.57 ± 12.55 % (Fig. 6), losing efficiency when the air flow rate was raised. Also, at 21st day the anammox activity had an ammonium and nitrite consume rate of 9.24 mgN L−1 h−1 and 10.90 mgN L−1 h−1 respectively (Table 3). It shows that anammox activity was lower from what was observed in the other phases and in the reactor inoculation. At 51st
Fig. 6 Stoichiometric coefficients during operation time in test 2 named study compared with Daverey et al. (2013)
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day, bacteria quantification showed that anammox bacteria had stable growth and nitrifying bacteria community increased 1 log10. (Table 3). The increase on aeration rate during this phase has produced a negative effect on deamonification process, once there was an excessive nitrifiers growth, which suppressed the activity of anammox bacteria. According to Zheng et al. (2013), expanded bed reactors are shown as a more efficient configuration to biomass retention. This may have contributed to the increasing growth of bacteria with anammox activity in the reactor. The control of operating parameters make possible to maintain the balance in the two bacterial populations in the reactor. Consequently, the nitrogen removal efficiency was not affected by this difference (Daverey et al. 2013).
4 Conclusion Low recirculation rate create better conditions to reactor activity, keeping anammox sludge in good shape and activity. High recirculation rate increased nitrifiers growth and created conditions to anammox floc disintegration and inhibition. Oxygen favor nitrifier activity and cause NO2–N accumulation inside the reactor and DO is a good tool to control deammonification process. Acknowledgements This study had financial support from CAPES, CNPq, and Eletrosul.
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