Effect of Free Ammonia, Free Nitrous Acid, and Alkalinity on the Partial Nitrification of Pretreated Pig Slurry, Using an Alternating Oxic/Anoxic SBR

Research Article Effect of Free Ammonia, Free Nitrous Acid, and Alkalinity on the Partial Nitrification of Pretreated Pig Slurry, Using an Alternating Oxic/Anoxic SBR Marisol Belmonte,1,2,3 Chia-Fang Hsieh,1José Luis Campos,4Lorna Guerrero,5 Ramón Méndez,6Anuska Mosquera-Corral,6and Gladys Vidal1 1Engineering and Environmental Biotechnology Group, Environmental Science Faculty & Center EULA-Chile, University of Concepci´ on,P.O.Box160-C,Concepci ´ on, Chile 2School of Biochemical Engineering, Pontificia Universidad Cat´ olica de Valpara´ ıso, 2362803 Valpara´ ıso, Chile 3Laboratory of Biotechnology, Environment and Engineering, Faculty of Engineering, University of Playa Ancha, 2340000 Valpara´ ıso, Chile 4Facultad de Ingenier´ ıa y Ciencias, Universidad Adolfo Ib´ a˜ nez, 2503500 Vi˜ na del Mar, Chile 5Department of Chemical and Environmental Engineering, University Federico Santa Mar´ ıa, 2390123 Valpara´ ıso, Chile 6Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Correspondence should be addressed to Marisol Belmonte; [email protected] Received 18 May 2017; Accepted 1 August 2017; Published 6 September 2017 Academic Editor: Giuseppe Olivieri Copyright © 2017 Marisol Belmonte et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The effect of free ammonia (NH3or FA), free nitrous acid (HNO2or FNA), and total alkalinity (TA) on the performance of a partial nitrification (PN) sequencing batch reactor (SBR) treating anaerobically pretreated pig slurry was studied. The SBR was operated under alternating oxic/anoxic (O/A) conditions and was fed during anoxic phases. This strategy allowed using organic matter to partially remove nitrite (NO2−)andnitrate(NO 3−) generated during oxic phases. The desired NH4+to NO2−ratioof1.3gN/gNwas obtained when an Ammonium Loading Rate (ALR) of 0.09 g NH4+-N/L⋅d was applied. The system was operated at a solid retention time (SRT) of 15–20 d and dissolved oxygen (DO) levels higher than 3 mg O2/L during the whole operational period. PN mainly occurred caused by the inhibitory effect of FNA on nitrite oxidizing bacteria (NOB). Once HNO2concentration was negligible, NH4+was fully oxidized to NO3−in spite of the presence of FA. The use of biomass acclimated to ammonium as inoculum avoided a possible effect of FA on NOB activity. 1. Introduction The intensive swine production is creating scenarios where generated waste is not correctly disposed, exceeding the assimilation capability of the soil-water-plant ecosystem of the crop lands [1]. The anaerobic digestion is the most used technology to treat this kind of wastes [2]. In this process, high removal efficiencies of carbonaceous compounds contained in the wastewater are achieved while nitrogen removal is scarce, only due to biomass growth. Since the effluent from the anaerobic digester has a low C/N ratio, to perform nitrogen removal by the combination of nitrification (sequential ammonium (NH4+) oxidation to nitrite (NO2−) and nitrate (NO3−)) and denitrification (nitrate or nitrite reduction to nitrogen gas (N2)) processes is not economically feasible due to the requirements of organic matter. The application of the combined partial nitrification (oxidation of ammonium to nitrite with around 50% efficiency) and anammox (combination of previously generated nitrite and ammonium to produce nitrogen gas) processes could avoid this drawback. However, some studies reflected problematic situations for nitrogen removal in this way due to the presence Hindawi BioMed Research International Volume 2017, Article ID 6571671, 7 pages https://doi.org/10.1155/2017/6571671 2BioMed Research International Aeration Stirring Feeding Settling Withdrawal Time (min) 105 5 55 105 5 55 105 5 55 105 5 55 45 15 Figure 1: Distribution of the operational cycle. of relevant concentrations of residual organic matter in the treated effluent [3]. In this sense, Wett et al. [4] proposed to treat a municipal wastewater, with a low C/N ratio, in a partial nitrification unit operated in alternated oxic and anoxicperiodsinordertopromotetheuseoftheorganic matter present for denitrification. This strategy together with the control of the solid retention time (SRT) also allowed suppressing the growth of nitrite oxidizers when the unit was operated at low temperature and low ammonium concentrations and, therefore, improving the stability of partial nitrification. Moreover, as the organic matter was removed by denitrification, alkalinity was generated which partially compensated for the alkalinity consumption due to partial nitrification. During the treatment of wastewater with high ammonium concentrations, as the effluent of pig slurry coming fromtheanaerobicdigestion,thepresenceoffreeammonia (NH3or FA) and/or free nitrous acid (HNO2or FNA) can affect the performance of the partial nitrification process. These compounds can cause inhibition of nitrifying and denitrifying bacteria and provoke the nitrite accumulation in the system [5, 6]. The nitrifying bacteria are inhibited at concentrations of FA and FNA within 0.1–150 mg NH3N/L and 0.2–2.8 mg HNO2-N/L, respectively [5], while the effect of FNA on denitrifying bacteria was observed within 0.01–0.20 mg HNO2-N/L [7]. Another factor to be considered is the inlet total alkalinity/ammonium ratio (TA/NH4+-N) since it will determine the pH value inside the reactor and, therefore, the concentrations of FNA and/or FA [8–10]. InthepresentresearchtheeffectofFA,FNA,and total alkalinity/ammonium ratio on the performance of a partial nitrification sequencing batch reactor (SBR) operated under alternating oxic/anoxic conditions was studied. An anaerobically pretreated pig slurry and acclimated biomass to high ammonium concentrations were used as feeding andinoculum,respectively.Theoperationalconditionswere adjusted to achieve the desired nitrite to ammonium ratio in the effluent and promote the consumption of the present organic matter by means of the denitrification process. 2. Materials and Methods 2.1. Reactor SBR Description and Operational Conditions. A laboratory scale SBR with a working volume of 1.5 L and a total volume of 2.5 L was used. Dimensions of the unit were height of 540 mm (𝐻), inner diameter of 77 mm (𝐷), and the 𝐻/𝐷 ratio of 7. Oxygen was supplied by means of a ceramic air diffuser located at the bottom of the reactor connected to an air pump. The system was equipped with a mechanical stirrer operated at 80 RPM. The reactor was maintained in a thermostated chamber at 33 ±2∘C. The pH was not controlled and ranged between 6.2 and 8.5. A programmable logic controller (PLC) was used to control the cycle. The reactor was operated in cycles of 12 h distributed as showninFigure1.Thevolumeexchangeratiowasfixedat 8.3% and the hydraulic retention time (HRT) was of 6 days. The DO was supplied only during the oxic period and its concentration was kept higher than 3 mg O2/L. In the anoxic phase the mixture inside the reactor was achieved through mechanical stirring. The reactor was fed with the effluent coming from an anaerobic digester treating diluted pig slurry [2], whose total alkalinity/NH4+ratio ranged from 4.0 to 9.4 g/g. The reactor was operated during 270 days divided into three stages according to the inlet ammonium concentrations of 350, 550, and 880 mg NH4+-N/L, which corresponded to applying Ammonium Loading Rates (ALRs) of 0.06, 0.09, and 0.15 g NH4+-N/L⋅d, respectively (Table 1). The SRT was not controlled and ranged from 15 to 20 d during the whole operational period. 2.2. Activity Assays. Periodical samples of biomass were collected from the reactor during the operational stages to evaluate their specific ammonium and nitrite oxidizing activities (AOB and NOB, resp.) and specific denitrifying activity (SDA). The specific nitrifying activity (ammonium and nitrite oxidizing) of the biomass was determined by respirometric assays, applying the methodology described by L´ opez-Fiuza et al. [11], while the maximum SDA of the sludge was determined according to the methodology proposed by Buys et al. [12]. 2.3. Inoculum. The SBR was inoculated with 5 g volatile suspended solids (VSS)/L of activated sludge collected from an aerobic reactor, used to remove both organic matter and nitrogen from pig slurry, located in the Region of the Libertador Bernardo O’Higgins, Chile. The initial specific BioMed Research International 3 Table 1: Characterization of the different operational stages of the SBR reactor. Parameter Unit Stage I II III Influent Effluent Influent Effluent Influent Effluent Operation time d 0–75 76–190 191–270 ALRs g NH4+-N/L⋅d 0.06 0.09 0.15 Total alkalinity/NH4+-N g/g 9.4± 0.0 —7.5 ±0.0 —4.1 ± 0.0∗— pH 7.5± 0.1 7.4 ±1.3 7.5± 0.1 6.8± 0.9 7.5± 0.1 7.2±1.3 CODSmg/L 734 ± 85 415 ± 28 801± 100 363 ±189 1907 ± 319 293± 58 NH4+-N mg/L 350± 26 82±25 550 ±67 128 ± 77 880± 100 102± 60 NO2−-N mg/L <1.0 174± 32 <1.0 165 ± 86∗<1.0 2±2 NO3−-N mg/L <1.0 45± 5 <1.0 42 ± 17∗<1.0 293 ± 138 NO2−-N/NH4+-N g/g — 2.1± 0.5 —1.3 ±0.5 —<0.1 ±<0.1 NIT % 79± 5 76 ± 5 86± 11 NAR % 78± 2 83 ±6∗∗ 1±<0.1 Nremoved %21 ± 13 30± 7 45± 19 CODremoved %39 ±9 54 ± 11 85± 3 CODremoved/Nremoved g/g 2.8 ±1 2.6 ± 0.4 3.5± 0.5 ∗Sodium bicarbonate (NaHCO3) was added to the influent to keep it at total alkalinity/NH4+-N ratio of 4.1 g/g. ∗∗During the stable period (days 76 to 160). nitrifying activities obtained for AOB and NOB were of 27 mg NH4+-N/g VSS⋅dand19mgNO 2−-N/g VSS⋅d, respectively, and the initial SDA was of 72 mg NO2−-N/g VSS⋅d. 2.4. Analytical Methods. Concentrations of soluble chemical oxygen demand (CODS), VSS, NH4+,NO 2−,andNO 3− were determined according to the Standard Methods [13]. Total alkalinity was determined by titration according to the methodology described by Ripley et al. [14]. The pH value and DO concentrations were measured using specific electrodes (pH: sensor Multiparameter Oakton PC650 model; DO: sensor Oxi 330 WTW). 2.5. Calculations. The nitrification (NIT) and nitrite accumulation ratio (NAR) percentages were calculated according to the following equations: NIT =NH4+-N𝑖−NH4+-N𝑒 NH4+-N𝑖⋅100 (1) NAR =NO2−-N𝑒 NO3−-N𝑒+NO2−-N𝑒⋅ 100, (2) where NH4+-N𝑖and NH4+-N𝑒are the concentrations of ammonia in the influent and effluent, respectively, and NO2−- N𝑒and NO3−-N𝑒are the concentrations of nitrite and nitrate in the effluent, respectively. The ratio between the amount of organic matter removed and nitrogen removed (CODremoved/Nremoved,gCOD S/g N) was calculated according to the following equation: CODremoved Nremoved =(COD𝑖−COD𝑒) NH4+-N𝑖−(NO3−-N𝑒+NO2−-N𝑒+NH4+-N𝑒). (3) The concentrations of NH3and HNO2were calculated from the NH4+-N and NO2−-N concentrations inside the reactor, respectively, at the operating temperature and the pH value in the bulk liquid according to the expressions proposed by Hou et al. [15]. Ammonium and nitrite consumption rates were calculated from the profiles of ammonium and nitrite concentrations obtained in the reactor from the measurements performed throughout the operating cycles as described by Mosquera-Corral et al. [16]. 3. Results and Discussion 3.1. Operation of the Reactor. The partial nitrification process was developed in the reactor after 25 days of operation (Stage I). The NAR, defined as the produced nitrite to ammonium removed ratio, was of 78% (Table 1). However, the achieved NO2−-N/NH4+-N ratio in the effluent (2.1 g/g) was higher than the required value of 1.3 g/g which is considered optimal for the posttreatment of the effluent by means of the anammox process [17, 18]. The very high nitrite production, with efficiencies larger than required, indicated the system was underloaded. On day 76, ALR was increased from 0.06 to 0.09 g NH4+-N/L⋅d (Stage II) in order to overload the system and, therefore, to obtain the desired NO2−-N/NH4+- N ratio. In these conditions the NAR remained almost constant (83%) while the ammonium level in the effluent increased which allows obtaining a NO2−-N/NH4+-N ratio in the effluent of 1.3 g/g. On day 160 (Stage II) a failure of the aeration system caused that the system operated under anoxic conditionsformorethan48handnitriteconcentrationin the reactor decreased from around 200 to 100 mg NO2−- N/L (Figure 2(a)). After this event the partial nitrification turned unstable and NAR diminished progressively while the nitrate concentration increased. In Stage III, the applied 4BioMed Research International Nitrogen concentration (mg/L) Stage I Stage II Stage III 50 100 150 200 250 3000 Time (d) 0 200 400 600 800 1000 1200 (a) Stages I and II Stage III 0 3 6 9 12 15 ．（3-N (mg/L) 20 40 60 80 1000 NAR (%) 0.0 0.1 0.2 0.3 0.4 0.5 Stage ）） → ））） 2-N (mg/L) HNO (b) Stages I and II Stage III Stage ）） → ））） 9.4 g/Ａ→7.57.5 g/Ａ→4.1g/g TA/．（4+-N inf: 4.1 g/g 0 2000 4000 6000 8000 10000 Total alkalinity (mg CaC／3/L) 20 40 60 80 1000 NAR (%) (c) Figure 2: Evolution of nitrogen compounds. (a) Behavior of nitrogen concentration inside the reactor: NH4+-N influent (diamond), NH4+-N effluent (square), NO2−-N effluent (triangle), and NO3−-N effluent (circle). (b) Nitrite accumulation ratios (NAR) as percentages obtained at different HNO2-N (asterisk) and NH3-N (circle) concentrations. (c) Nitrite accumulation ratios (NAR) as percentages obtained at different total alkalinity influent (diamond) concentrations. 0 500 1000 1500 2000 ＃／＄Ｌ？ＧＩＰ？＞ (mg CODs/L) 2500 200 400 600 8000 NＬ？ＧＩＰ？＞ (mg/L) Figure 3: Relationship between CODSand N removed throughout the operational period. ALR was increased up to 0.15 g NH4+-N/L⋅dwiththeaim of overloading the nitrifying microorganisms and, therefore, of accumulating nitrite, but the system was able to fully oxidize ammonium to nitrate at an efficiency of 86% and the previous partial nitrification conditions were not recovered. The destabilization of the PN that occurred on day 160 couldberelatedtotheprolongedanoxicperiodsufferedby both AOB and NOB due the failure of the aeration system. According to Tor` a et al. [19], the activity of AOB would decrease around 14% after two days under anoxic conditions but the decrease expected for NOB activity would be higher than that of AOB activity due to the higher value of their decay constant [20]. Then, the effect the prolonged anoxic period on the loss of PN stability could be attributed to the decrease of nitrite concentration observed during this period. In order to evaluate the possible reasons for the loss of nitrite accumulation, the FA and FNA concentrations were estimated for the different operational stages as shown in Figure 2(b). Collected results indicate that high NAR were obtained in the presence of FNA concentrations within 0.08–0.37 mg HNO2-N/L. These concentrations could be responsible for the inhibition of NOB and the consequent nitrite accumulation as it has been observed by authors like Park and Bae [21] and Kim and Seo [22]. On the contrary, no correlation between nitrite accumulation and FA concentrations calculated was detected. Ammonium was fully oxidized to nitrate even at FA concentrations higher than 3 mg NH3-N/L, a value which is generally reported as inhibitory for NOB [5, 21, 23]. Nevertheless, if biomass is adapted to the FA, the NOB inhibition can be only achieved at FA concentrations higher than 10 mg NH3-N/L [24–26]. BioMed Research International 5 Oxic Anoxic Oxic Anoxic Oxic Anoxic Oxic Anoxic CODs (g) 246810120 Time (h) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 ．（4+,．／ 2−,．／ 3−(g N) 0.00 0.20 0.40 0.60 0.80 1.00 (a) CODs (g) Oxic Anoxic Oxic Anoxic Oxic Anoxic Oxic Anoxic 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 ．（4+,．／ 2−,．／ 3−(Ａ N) 0.00 0.20 0.40 0.60 0.80 1.00 1.20 246810120 Time (h) (b) Figure 4: Evolution of CODS(circle), NH4+-N (square), NO2−-N (triangle), and NO3−-N (diamond) calculated amounts inside the reactor during an operational cycle in the SBR on days 109 (Stage II) (a) and 221 (Stage III) (b). ResultsindicatethatNARwasquicklylostoncetheinhibitory conditions for NOB were suppressed in Stage III. This would demonstrate that the inhibitory effects of FNA on NOB were temporary [15, 27]. Alkalinity is another important parameter that affects the nitrification process since it is important to maintain a suitable pH value [28]. Figure 2(c) shows that the decrease of the inlet total alkalinity concentrations increased the efficiency of the system in accumulating nitrite. This can be attributedtothedecreaseofpHvalueinsidethereactor, which promoted the inhibitory effect of FNA on NOB (see Table 1). On the contrary, Hwang et al. [10] and Hou et al. [15] 6BioMed Research International observed that nitrite was only accumulated when the inlet TA/NH4+-N ratio was higher than the stoichiometric one (7.1 g TA/g NH4+-N) since this favoured the FA. Respirometric assays were performed with biomass samples from the reactor. Experimental results confirmed the observations from the reactor operation. The initial NOB specific activity decreased to no detectable levels on day 25 and remained at this level until day 187. These results were related to the increase in the FNA concentration and its inhibitory effect on NOB (Figure 2(b)). From this day on, NOB specific activity gradually increased up to a value of 230 mg NO2−-N/g VSS⋅d. The AOB specific activity was of 47, 66, and 124 mg NH4+-N/g VSS⋅d during Stages I, II, and III, respectively. The SDA of the biomass was of 120 mg N/g VSS⋅d during the whole operational period. The COD removal efficiency was affected by the influent quality and ranged from 39% during Stage I to 85% during Stage III. Concerning nitrogen removal efficiency, its value ranged from 21% (Stage I) to 47% (Stage III) (Table 1). Results showed a clear relationship between the amounts of organic matter and nitrogen removed, which indicate that the denitrification process was taking place (Figure 3). During Stage II, the CODremoved/Nremoved ratio was of 2.6 g/g while, during Stage III, this ratio increased up to 3.5 g/g because ammonium was completely oxidized to nitrate during this stage [29]. 3.2. Operational Cycles. In order to evaluate the performance of the reactor, cycle measurements were carried out. The CODSand nitrogenous compounds concentrations were measured throughout different operational cycles over the operational period. Data obtained from the cycle measurements of days 109 (Stage II) and 221 (Stage III) are shown in Figure 4 in terms of accumulative amounts (g) ofthemeasuredcompounds.Inbothcycles,biodegradable organic matter was mainly removed during the anoxic phases together with nitrite and nitrate while CODSremained practically constant during oxic phases. These profiles show that the cycle distribution allows the efficient use of the organic matter, contained in the influent, to carry out denitrification. Therefore, the nitrogen load to be treated in a subsequent anammox reactor decreases. The main difference between both measured cycles was the relative values of ammonium and nitrite oxidation rates. On day 109, the ammonium oxidationratewas3to4timeslargerthanthenitriteoxidation rate, while on day 229, both values were almost the same thus indicating that the partial nitrification was not possible during the last stage. Gilbertetal.[30]observedtheappearanceofalag phase longer for the nitrite oxidation than for the ammonia oxidation when anoxic periods, longer than 15–20 minutes, were imposed. However the lag phase corresponding to the nitrite oxidation had been always lower than 12 minutes. Therefore, the operational cycle distribution used in this work would not be suitable to promote PN due to the long duration of the oxic period (105 minutes). In fact, when alternating oxic/anoxic conditions are applied to avoid the nitrite oxidation during municipal wastewater treatment by means of continuous systems, where neither FA nor FNA inhibitions are expected, the length of the oxic and anoxic periods is fixed at around 10 minutes [4, 29, 31]. 4. Conclusions Stable partial nitrification was achieved during the treatment of the effluent of an anaerobic reactor fed with pig slurry. Since the system was operated at high STR values (15–20 d) and nonlimiting oxygen concentrations (higher than 3 mg O2/L), nitrite accumulation was mainly attributed to the presence of high FNA concentrations. Attempts to inhibit NOB by promoting the presence of FA were not successful. Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication and financing the publication of this paper. Acknowledgments This work was funded by the Chilean Government (CONICYT/FONDECYT 3140276, CONICYT/PAI/MEC 80140011, FDD-UPLA-CC18105, Patagonia Seed Project 210.310.055-1SP, CONICYT/FONDAP/15130015, and Red Doctoral REDOC.CTA, MINEDUC Grant UCO1202 at University of Concepci´ on). The authors thank Mr. C. Contreras from “Sucesi´ on Yanine Abadi” for allowing the realization of this study in their facilities. References [1] B. Fridrich, D. Krˇ cmar, B. Dalmacija et al., “Impact of wastewaterfrompigfarmlagoonsonthequalityoflocalgroundwater,” Agricultural Water Management,vol.135,pp.40–53,2014. [2] D.C.Rodriguez,M.Belmonte,G.Pe ˜ nuela, J. L. Campos, and G. Vidal, “Behaviour of molecular weight distribution for the liquid fraction of pig slurry treated by anaerobic digestion,” Environmental Technology,vol.32,no.4,pp.419–425,2011. [3] S. 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