Journal of Bioscience and Bioengineering VOL xx No xx, 1e8, 2016 www.elsevier.com/locate/jbiosc Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier Thanh Tung Vo and Tan Phong Nguyen* Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, VietNam Received 17 June 2015; accepted January 2016 Available online xxx Single-stage nitrogen removal using Anammox and partial nitritation (SNAP) is a novel technology developed in recent years for removing nitrogen To evaluate the ability of SNAP technology to remove nitrogen in old landfill leachate under the conditions in Vietnam, we conducted a survey with different nitrogen loading rates of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 kg-N/m3 day and a concentration from 100 to 700 mg-N/L The operating conditions were as follows: DO at 1.0e5.3 mg/L, HRT at 12 h, and pH at 7.5e7.8 The biomass carrier was a biofix made from acrylic fiber The maximum ammonium conversion and nitrogen removal efficiency were approximately 98% and 85%, respectively, at 1.2 kg-N/ m3 day In general, the nitrogen removal efficiency increased and stabilized at the end of each loading rate The first step showed that SNAP could potentially be applied in real life for removing nitrogen from old landfill leachate Ó 2016, The Society for Biotechnology, Japan All rights reserved [Key words: SNAP; Anammox; Nitritation; Old landfill leachate; Biofix] Leachate is generated from landfills of municipal solid waste and is a major concern for the surrounding environment Its negative impacts include damaging the receiving sources if it has not been thoroughly treated In particular, the nitrogen concentration in leachate is relatively high Therefore, the problem of treating nitrogen in the leachate is a subject concerning scientists in particular and each nation in general Conventional nitrification/denitrification technologies are not suited for treating old landfill leachate with high ammonium nitrogen concentrations and low biodegradable organic matter content (1) Traditional technologies require additional external carbon sources and large energy consumption and so on Thus, applied research has been conducted on individual or combined partial nitritation and anammox processes to reduce operating costs and ensure the nitrogen processing efficiency with a high nitrogen loading rate, such as single reactor system for high ammonium removal over nitrite (SHARON-Anammox) (2), completely autotrophic nitrogen removal over nitrite (CANON) (3), oxygen-limited autotrophic nitrification-denitrification (OLAND) (4), and single-stage nitrogen removal using Anammox and partial nitritation (SNAP) (5) Compared with conventional nitrification/denitrification technologies, partial nitritation/anammox reduced 85% of the oxygen requirement, 100% of the carbon requirement and 83% of the bio-solid production (6) The SNAP process is based on two biotechnologies In the first, partial nitritation, ammonium is partly nitrified to nitrite by ammonia-oxidizing bacteria (AOB) (Eq 1) (5), and then the * Corresponding author Tel./fax: ỵ44 38639682 E-mail address: ntphong@hcmut.edu.vn (T.P Nguyen) resulting nitrite is denitrified with the residual ammonium in the Anammox (Eq 2) (7) ỵ þ 2NHþ þ1:5O2 /1NH4 þ1NO2 þ1H2 O þ 2H (1) 1NHỵ ỵ1:146NO2 /0:986N2 ỵ0:161NO3 ỵ2H2 O (2) The overall reaction is described in Eqs and 5: ỵ 1NHỵ ỵ 0:85O2 /0:44N2 ỵ 0:11NO3 þ 1:43H2 O þ 1:14H (3) In a few recent studies on SNAP, Lieu et al (8) performed more research on SNAP technology with a landfill leachate with low ammonium nitrogen Takekawa et al (9) studied the effects of the operational conditions of the SNAP process Hien et al (10) evaluated the nitrogen removal efficiency from synthesized wastewater with a high nitrogen concentration using SNAP technology Further research is required on SNAP technology for the treatment of real landfill leachate with high ammonium nitrogen concentrations to create a precondition for the application of this technology in practice under conditions in Vietnam MATERIALS AND METHODS Experimental set-up and operational conditions The SNAP reactor design features a cylinder with a conical bottom made of acrylic resin and a working volume of 6.5 L Its internal diameter and useful height tank are 150 mm and 420 mm, respectively The schematic diagram of the experiment is shown in Fig The biomass carrier used in this research is biofix, which is made from a hydrophilic net-type acryl resin fiber material (NET Co., Ltd., Hyogo, Japan) The characteristics of this biomass carrier are shown in Table and Fig The biofix used had a weight of 41.2 g and was fixed by cylindrical frames with an external diameter of 92 mm, inner diameter of 62 mm and height of 300 mm The reactor operated with uncontrolled temperature and depended entirely on the ambient temperature, which ranged from 27 C to 35 C Air was fed into the 1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan All rights reserved http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VO AND NGUYEN J BIOSCI BIOENG., Influent tank Air-co ntrolling valve NaHCO3 solution Reac tor Influent pump Biomass carriers NaHCO3 pump 10 Dis charge valve sludge pH controller 11 Cen ter resin tube Air pump FIG Schematic diagram of experimental apparatus reactor through the bottom of the center tube and controlled by an air-controlling valve such that the dissolved oxygen (DO) did not exceed mg/L The DO increased gradually with increasing nitrogen loading rate The pH and hydraulic retention time (HRT) were suitable for SNAP, pH 7.5e7.8 and 12 h, respectively (5) NaHCO3 solution (0.5 N) was used to control the pH value through a pH controller In addition, to achieve partial nitritation and avoid the production of nitrate ðNỒ À NÞ, the operation required certain conditions, such as a low dissolved oxygen concentration (DO) (11e13), high temperature (14), pH (15) and free ammonia concentration (FA) (16) The DO was strictly controlled, as mentioned above, with a measurement frequency of times/day FA was controlled through the adjustment of the ammonium concentration and pH in the tank During the Tet holiday, the research was maintained with the operating condition of an NLR of 0.6 kg-N/m3 day À NH3-B Preliminary Distillation Step, NOÀ À N : 4500ÀNO2 À B Colorimetric À Method, and NOÀ À N : 4500ÀNO3 À E Cadmium Reduction Method (17) The total nitrogen was defined by TCVN 6638:2000 (National Technical Regulation Vietnam), Catalytic digestion-devarda alloy (18) The pH was controlled by pH controller BL 931700, HannaeEngland DO was measured using EXTECH 407510eTaiwan Polymerase chain reactionePCR Polymerase chain reaction (PCR) analysis was conducted to confirm the existence and identification of nitrifying and anammox bacteria in the SNAP sludge The entire analytical process was conducted by the Seed sludge and influent wastewater The anammox sludge and the activated sludge seeding in the reactor were g/L and g/L, respectively (5) They were mixed into the SNAP sludge The total seed sludge was g/L (MLSS) The anammox sludge and activated sludge were obtained from Kumamoto University, Japan, and the Tan Binh wastewater treatment plant, Vietnam, respectively In this study, a synthetic landfill leachate simulating pretreated leachate was used as the influent for the start-up phase for the SNAP sludge Then, the synthetic leachate and old leachate were mixed together at different ratios (the ratios of synthetic leachate and old leachate were two weeks each of 8:2, 6:4, 4:6, and 2:8) until the old landfill leachate was completely replaced at 0.4 kg-N/m3 day When real landfill leachate was used in the experiment, the nitrogen concentration in the influent wastewater increased to achieve nitritation and the SNAP process The composition of the synthetic and old landfill leachate are shown in Tables and 3, respectively Chemical analysis Standard Methods for the Examination of Water and Wastewater (USA) were used to analyze ammonium-nitrogen NHỵ Nị, nitrite ỵ nitrogen NO NÞ and nitrate-nitrogen ðNO3 À NÞ, including NH4 À N: 4500- TABLE Properties of acryl-fiber biomass carrier Valuea Parameter Specific yarn length (m/m ) Specific surface area (m2/m3) Yarn diameter (mm) a Provided by the manufacturer 23.324 146.5 FIG Photos of biomass carriers and biomass carrier fixed on steel frames Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VOL xx, 2016 NITROGEN REMOVAL FROM LEACHATE WITH SNAP TECHNOLOGY RESULTS AND DISCUSSION TABLE Composition of synthetic landfill leachate Composition Concentration NH4Cl (mg-N/L) NaHCO3 (mg/L) KHCO3 (mg/L) KH2PO4 (mg/L) MgSO4$7H2O (mg/L) CaCl2$2H2O (mg/L) FeSO4$7H2O (mg/L) Na2$EDTA (mg/L) KHPa (mg/L) Na2SO3b 100e200 160 160 43 328 235 16 16 38 160 Potassium hydrogen phthalate (C8H5O4K) Controlling the DO in influent of synthetic wastewater TABLE Eater quality of old leachate from the Go Cat landfill Constituent Value pH Alkalinity (mg/L) TKN (mg/L) NH4-N (mg/L) NO2-N (mg/L) NO3-N (mg/L) COD (mg/L) BOD5 (mg/L) Total phosphate (mg/L) Total hardness (mg CaCO3/L) SO2À (mg/L) ClÀ (mg/L) Total iron (mg/L) SS (mg/L) 8e8.5 15.133 3.868 3449 0.2052 2.22 2761 50 20.9 15.567 0.9472 3442 2.16 59 Ỉ Ỉ Ỉ Ỉ Ỉ Ỉ Ỉ Ỉ Ỉ Æ Æ Æ Æ 58 26 233 0.01 0.18 436 20 2.9 25 0.08 26 0.07 16 Adopted from Biec (30) Institute of Tropical Biology Analytical procedures including DNA separation, DNA amplification with special oligonucleotide primers, and 16S rDNA sequencing of bacteria by the Macrogen Company, Korea, and then the retrieved gene sequences were compared with homologous genes using BLAST with NCBI databases The special oligonucleotide primers for anammox are Ana-50 (50 -TAGAGGGGTTTTGATTAT-30 ) and Ana-30 (50 -GGACTGGATACCGATCGT-30 ) (19) pH I DO II Temperature III IV V VI 37 Tet holiday Variations in pH, DO, and temperature As shown in Fig 3, the pH value was controlled in the appropriate range for the SNAP process by an automatic pH controller with the starting pH value of 7.7 (5) The pH greatly affected two species of AOB and anammox bacteria in SNAP because it was the essential factor for determining the free ammonia (FA) and free nitric acid (FNA) concentration in the tank FA and FNA were two inhibitors for the nitrification process in general and the partial nitritation process in particular The concentration of FNA was generally lower than the nitrifier-inhibited values of 0.2e2.8 mg/L (16) Hence, FA was consulted more because it was also a contributing factor to the inhibition of unwanted nitrite oxidizing bacteria (NOB) in SNAP with an inhibitive range of FA for NOB and AOB were reported to be 0.08e0.82 mg/L and 10e150 mg/L, respectively (16) The concentrations of FA and FNA corresponding to the nitrogen loading rate are shown in Table DO was the most important parameter of the SNAP process If the DO was too low or too high, it would directly affect the activity of AOB and anammox bacteria Therefore, the DO must be adjusted to optimize partial nitritation reaction and not to inhibit anammox reaction simultaneously DO was controlled based on the analytical results of the effluent nitrite and nitrate concentrations The DO increased from 1.0 to 4.8 mg/L with increase in NLR when the microorganisms stabilized and increased the biomass The DO obtained in this study was slightly lower than the reported values by Takekawa et al (9) and Qiao et al (20) This DO value is higher than those in the studies by Lieu et al (8) and Hien et al (10) because the SNAP reactor was operated under a high loading rate using real landfill leachate as influent and used real landfill leachate Therefore, a higher DO supply was necessary to oxidize a number of other components in the landfill leachate In addition, temperature was also a notable condition during the experiment because it partly contributed to selecting AOB and eliminating NOB However, the temperature was not controlled and 35 pH, DO (mg/l) 33 31 29 Temperature (0C) a b 27 25 50 100 I - 0.2 kg-N/m3.day II - 0.4 kg-N/m3.day III - 0.6 kg-N/m3.day 150 200 Time (days) 250 300 IV - 0.8 kg-N/m3.day V - 1.0 kg-N/m3.day VI - 1.2 kg-N/m3.day FIG Changes in pH, DO, and temperature in the reactor Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VO AND NGUYEN J BIOSCI BIOENG., bacterial activity (15) The inhibition of AOB indicated that partial nitritation had not occurred as a result and the anammox bacteria could not participate in the reaction of SNAP reactor Similar to the ACE results, the nitrogen removal efficiency (NRE) decreased strongly at the beginning of each loading rate Consequently, the FA concentration must be controlled at an appropriate value in the tank via pH The AOB activity recovered in a timely manner and converted ammonium well again Also, high NRE of 83.1%, 83.7% and 85.2% were obtained on the last day in the last three runs Highest and stable NRE with a standard deviation of Ỉ6.03% was obtained under NLR of 1.2 kg-N/m3 day This suggested favorable and stable microbial growth was obtained in SNAP reactor Fig shows that the average values of ACE and NRE gradually increased stably over the loading rates The average ACEs for six runs were 55.6%, 58.0%, 60.4%, 76.6%, 79.1% and 83.8% Similar to ACE, NRE also gradually increased to 46.4%, 51.8%, 53.6%, 69.6%, 69.1% and 73.5% under the six loading rates At 1.0 kg-N/m3 day, the NRE value did not increase and was close to the value of the previous loading rate The reason for that was the withdrawal of sediment which reduced the probability of water circulation and obstructed exposure of the substrates and microorganisms in the tank at 1.0 kg-N/m3 day Additionally, under this loading rate ACE increased only slightly compared with the other loading rates This was consistent with the theory that the AOB growth rate was faster than the anammox growth rate; therefore, ACE increased and NRE did not change compared with the 0.8 kg-N/m3 day loading rate On the other hand, the standard deviation of ACE and NRE also decreased significantly over the loadings This deviation decreased from Ỉ19.1% and Ỉ17.5% to Ỉ7.7% and Ỉ6.0%, respectively, with ACE and NRE at 1.2 kg-N/m3 day This indicates that the SNAP reactor experienced relatively good activity and stability over the time of operation The changes in the average values and standard deviations of ACE and NRE over the loading rates proved that an TABLE FA and FNA over loading rates Run NLR (kg-N/m3 day) I II III IV V VI VII VIII IXe 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.68 2.1 FNA (10À3 mg/L) FA (mg/L) 0.33 4.07 5.9 6.05 6.88 6.29 4.38 11.98 28.31 Ỉ Æ Æ Æ Æ Æ Æ Æ Æ 0.17 1.66 2.69 3.73 4.27 3.35 2.64 2.12 3.52 0.65 0.32 0.27 0.34 0.29 0.58 0.68 1.11 1.15 Ỉ Ỉ Ỉ Ỉ Æ Æ Æ Æ Æ 0.27 0.12 0.11 0.17 0.14 0.22 0.23 0.56 0.31 entirely depended on the ambient temperature As shown in Fig 3, it was recorded in the range between 27 and 35 C during this experiment This temperature range was very suitable for the growth of AOB and anammox bacteria (21,22) In addition, it also restricted NOB bacterial growth because AOB has a higher specific growth rate than NOB under temperatures above 25 C (23) Ammonium conversion, nitrogen removal efficiency and effluent nitrate nitrogen As shown in Fig 4, the ammonium conversion efficiency (ACE) increased and stabilized at the end of each loading rate In the loading rate of 0.2 kg-N/m3 day (run I), ACE rapidly achieved a high efficiency of approximately 88.6% on day 55 In the second loading (run II), ACE slightly decreased and had the highest value of approximately 71.4% Then, it also increased in the following loading rate (run III) This could be because the bacteria gradually stabilized and grew well In the last three loading rates (run IVeVI), ACE exceeded 90% regularly at the end of each run The highest ACE was recorded at the end of the 1.0 kg-N/m3 day loading rate, 98.2% on the 250th day Furthermore, ACE highly decreased at the beginning of each loading rate because the ammonium nitrogen concentration increased abruptly and the higher FA concentration inhibited AOB NH4+ I ACE II III NRE IV V VI 100 60 40 20 Nitrogen removal efficiency (%) 80 Tet holiday Nitrogen concentration (dg/L) 0 50 100 I - 0.2 kg-N/m3.day II - 0.4 kg-N/m3.day III - 0.6 kg-N/m3.day 150 200 IV - 0.8 kg-N/m3.day V - 1.0 kg-N/m3.day VI - 1.2 kg-N/m3.day 250 300 Time (days) FIG Variations in ammonium conversion and nitrogen removal efficiency and effluent nitrate concentration Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VOL xx, 2016 ACE NRE 0.02 and 0.01 As mentioned above, the denitrifier was limited in the early period, but the viability of the denitrifier was still possible in the tank During thickening, the biofilm created anoxic conditions for denitrifier activity Therefore, at the three last þ loading rates, the NOÀ À N=NH4 removal À N decreased significantly and showed a reduction of the effluent NOÀ À N concentration This observation was similar to the previous SNAP research of Lieu et al (5) and Hien et al (10) Referring to the study of the partial nitritation and anammox process, the nitrate reduction was also noted by Ganigue et al (24), Wang et al (25), and Yamagiwa et al (26) This contributes to increasing the treatment efficiency of the SNAP process NO3 eff 100 90 80 70 60 50 40 30 20 Effect of hydraulic retention time After surveying the treatment capabilities of SNAP through different loading rates under the conditions examined in previous studies of SNAP (5,9,10), we found that the loading changes were based on increasing the influent ammonium nitrogen concentration The results showed that the nitrogen removal efficiency of the SNAP model was very high Thus, SNAP technology may be applied in practice The research continued to study the more efficient removal of nitrogen with SNAP with higher loading rates through changes in hydraulic retention time (HRT) During the period of the HRT survey, the experimental conditions were maintained at NHỵ ẳ 700 ặ 3.6 mg/L, reducing HRT from 12 h to h as shown in Fig The operational time for each HRT stage was 30 days, similar to the time of the previous stages Observations showed that the ammonium conversion and nitrogen removal efficiency tended to decrease Specifically, at HRT of 12 h corresponding to 1.4 kg-N/m3 day, ACE and NRE increased and stabilized similarly to the stages of surveying treatment efficiency of the previous SNAP They ranged from 79 to 98% and 70e85%, respectively The DO was recorded at approximately 5.3 Ỉ 0.2 mg/L, and this value was maintained throughout the HRT survey At the end of this stage, the biomass in the SNAP reactor was measured, requiring a treatment efficiency stabilization period of approximately 10 days Then, other HRTs were surveyed However, after HRT was reduced by 10 h, ACE and NCE changed strongly, from 98% to 60% and from 89% to 52%, respectively 10 I II III kg-N/m3.day IV V VI Stage kg-N/m3.day I - 0.2 II - 0.4 kg-N/m3.day III - 0.6 kg-N/m3.day IV - 0.8 V - 1.0 kg-N/m3.day VI - 1.2 kg-N/m3.day FIG Average ammonium conversion and nitrogen removal efficiency and effluent nitrate concentration adequate bacterial density and status existed to perform the conversion process in the tank Additionally, according to Fig 5, the effluent NOÀ À N concentrations increased for the first three runs and tended to decrease for the subsequent loading rates In run I, the SNAP reactor used synthetic wastewater with a little organic matter according to the autotrophic bacteria activity of the nitrifier and anammox Furthermore, the operating conditions were maintained with a DO content of mg/L This limited the amount of denitrifier; thus, increasing the NOÀ À N concentration during first three run I to III may have caused (i) the byproduct of the anammox process or (ii) NOB partially converting nitrite to nitrate ỵ In run I, NO À N=NH4 removal À N ¼ 0:117, which was nearly equal to the reported reaction ratio of SNAP process Then, this ratio decreased in the subsequent loading rates to 0.09, 0.08, 0.04, ACE NRE HRT = 12 h HRT = 10 h NLR HRT = h HRT = 12 h 2.5 100 80 Period of measuring biomass Nitrogen removal efficiency (%) 60 40 20 280 300 320 1.5 NLR (kg-N/m3.day) Value (%) NITROGEN REMOVAL FROM LEACHATE WITH SNAP TECHNOLOGY 0.5 340 360 380 400 Time(days) I - 0.2 kg-N/m3.day II - 0.4 kg-N/m3.day III - 0.6 kg-N/m3.day IV - 0.8 kg-N/m3.day V - 1.0 kg-N/m3.day VI - 1.2 kg-N/m3.day FIG Effect of HRT on SNAP treatment performance Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VO AND NGUYEN Afterward, they increased gradually and stabilized in the range of 70 Ỉ 5% and 57 Ỉ 3%, respectively In the last stage of HRT ¼ 10 h, the processing efficiency was partly reduced by the FA concentration always exceeding the AOB inhibition of bacteria; thus, ACE was low during this period At HRT ¼ h, ACE and NRE clearly decreased through the dates of operation By the 15th day under the HRT of h, the ammonium conversion efficiency and nitrogen removal efficiency were 44% and 38%, respectively This result is similar to previous studies by Lieu et al (5) The concentration of suspended biomass increased This suggests that the AOB and anammox microorganisms were seriously inhibited and that the SNAP treatment capacity was decreased This was because HRT was not sufficient for the nitritation process to occur completely Thus, residual ammonium concentration increased highly and FA exceeded the AOB inhibition threshold constantly Moreover, the pH of landfill leachate was slightly high in the range of 8e8.5, and the ambient temperature was approximately 31e33 C Hence, FA was three times higher than the normal value and inhibited the nitrifier Through this study, the authors noted that the period in which the microorganism system in the SNAP model was completely inhibited was over 45 days The treatment capacity of SNAP required over 30 days for recovering and achieving a stable NRE in the range of 70%e75% J BIOSCI BIOENG., under HRT of 12 h The NRE values were slightly lower than before HRT was changed This was understandable because the anammox biomass was lost in the stage of complete inhibition; it needed more time to grow again In general, the research approach of surveying the capacity of the nitrogen treatment with high loading rates was suitable for increasing the influent nitrogen concentration On the other hand, the optimal hydraulic retention time for the SNAP process was still 12 h as the recommendation by Lieu et al (5) Observation of morphologic sludge Observed through naked eyes as shown in Fig 7, the authors found that the SNAP sludge changed gradually from brown-yellow to venetian red over operational stages This indicated that anammox bacteria grew well in the sludge attached on the biomass carrier because it is the characteristic color of anammox bacteria As mentioned above, the change in nitrogen loading rate depends on the increase in influent ammonium nitrogen concentration Thus, the leachate was diluted to the proper concentration for the study At initial loadings, the biomass color was observed relatively easily because of the high dilution ratio During the last loadings, color observation of biomass was low and the liquid color was more fuscous in the reactor FIG Changes in SNAP sludge color over the experimental period (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article) Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VOL xx, 2016 NITROGEN REMOVAL FROM LEACHATE WITH SNAP TECHNOLOGY ACKNOWLEDGMENT The authors wish to thank the laboratory staff of the Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, for their support throughout this study References FIG Electrophoresis profile of PCR-amplified DNA fragments TABLE Result of sequence analysis Species identified Anaerobic ammonium oxidizing planctomycete KOLL2a Uncultured anoxic sludge bacterium KU2 Candidatus Kuenenia stuttgartiensis (Anoxic biofilm clone Pla1-1) Uncultured anoxic sludge bacteria KU1 Similarity (%) NCBI No Reference 97 AJ250882 27 97 AB054007 28 97 AF202660 29 96 AB054006 28 In particular, the amount of biomass was relatively excessive and covered over the wall tank at loading of 1.4 kg-N/m3 day Therefore, could not observe the sludge color or any productive gas bubbles on the biomass carrier However, at the previous loading of 1.2 kg-N/ m3 day, the sludge nuance partly showed stability and the sludge attachment was active They exhibited good symbiosis together For example, ACE and NRE were 97% and 85%, respectively, at the NLR of 1.2 kg-N/m3 day Moreover, microbial consortia analyses were carried out on day 232 with stable MRE under NLR of 1.0 kg-N/ m3 day The result of the PCR shows clear bands approximately 200 base pairs (bp), and the special primers for anammox are Ana-50 (50 TAGAGGGGTTTTGATTAT-30 ) and Ana-30 (50 -GGACTGGA0 TACCGATCGT-3 ) (Fig 8) The result of the sequence analysis confirms the primers of many well-known anammox species, as shown in Table In further studies, we will progress towards the identification of AOB, denitrifiers and other autotrophic denitrifiers in the SNAP reactor Conclusion The SNAP reactor required a rather long time for adaptation and stabilization SNAP technology was capable of removing ammonium nitrogen under high NLR with closely controlled condition, such as pH 7.5e7.8, an optimal HRT of 12 h, and increasing DO with increasing nitrogen loading corresponding to 5.3 mg/L at 1.4 kg-N/m3 day The temperature conditions in the study area were suitable for the efficient performance of SNAP The maximum ammonium conversion and nitrogen removal efficiency were approximately 98% and 85%, respectively, at 1.2 kgN/m3 day This study shows that the denitrification process may still occur along with the anammox process It contributes to increasing the processing performance of SNAP In summary, the ammonium nitrogen treatment ability of SNAP technology has been verified, and it is completely applicable to the real treatment of old landfill leachate in practice under the conditions in Vietnam Kulikowska, D and Klimiuk, E.: The effect of landfill age on municipal leachate composition, Bioresour Technol., 99, 5981e5985 (2008) Van Dongen, U., Jetten, M S M., and Van Loosdrecht, M C M.: The SHARONAnammox process for treatment of ammonium rich wastewater, Water Sci Technol., 44, 153e160 (2001) Slierkers, A O., Derwort, N., Gomez, l L C., Strous, M., Kuenen, J G., and Jetten, M S M.: Completely autotrophic nitrogen removal over nitrite in one single reactor, Water Res., 36, 2475e2482 (2002) Pynaert, K., Wyffels, S., Sprengers, R., Boeckx, P., van Cleemput, O., and Verstraete, W.: Oxygen-limited nitrogen removal in a lab-scale rotating biological contactor treating an ammonium-rich wastewater, Water Sci Technol., 45, 357e363 (2002) Lieu, P K., Hatozaki, R., Homan, H., and Furukawa, K.: Single-stage Nitrogen Removal Using Anammox and Partial Nitritation (SNAP) for treatment of synthetic landfill leachate, Jpn J Water Treat Biol., 41, 103e112 (2005) Daigger, G T., Sanjines, P., Pallansch, K., Sizemore, J., and Wett, B.: Implementation of a full-scale anammox-based facility to treat an anaerobic digestion sidestream at the Alexandria Sanitation Authority Water Resource Facility, Water Pract Technol., http://dx.doi.org/10.2166/wpt.2011.033 (2011) Lotti, T., Kleerebezem, R., Lubello, C., and van Loosdrecht, M.: Physiological and kinetic characterization of a suspended cell anammox culture, Water Res., 60, 1e14 (2014) Lieu, P K and Chung, D T.: Lab-scale application of combined partial nitritation e anammox process for nitrogen removal from landfill leachate, Vietnam, J Sci Technol., 45, 99e101 (2011) Takekawa, M., Ohta, S., Yoshida, D., Sato, T., Kaneshiro, K., Soda, S., Ike, M., and Furukawa, K.: Effects of operational conditions on treatment performances of sing-stage nitrogen removal using anammox and partial nitritation process, Jpn J Water Treat Biol., 49, 133e142 (2013) 10 Hien, H T and Phong, T N.: Nitrogen removal from synthesis wastewater which high nitrogen concentration by single-stage using anammox and partial nitritation (SNAP) with BioFix as biomass carrier, Int J Earth Sci Eng (IJEE), 6, No 4, 727e731 (2013) 11 Wang, J L and Yang, N.: Partial nitrification under limited dissolved oxygen conditions, Process Biochem., 39, 1223e1229 (2004) 12 Ciudad, G., Rubilar, O., Muñoz, P., Ruiz, G., Chamy, R., Vergara, C., and Jeison, D.: Partial nitrification of high ammonia concentration wastewater as a part of a shortcut biological nitrogen removal process, Process Biochem., 40, 1715e1719 (2005) 13 Yang, J., Zhang, L., Daisuke, H., Takahiro, S., Ma, Y., Li, Z., and Furukawa, K.: High rate partial nitrification treatment of reject wastewater, J Biosci Bioeng., 110, 436e440 (2010) 14 Hellinga, C., Schellen, A A J C., Mulder, J W., van Loosdrecht, M C M., and Heijnen, J J.: The Sharon process: an innovative method for nitrogen removal from ammonium-rich waste water, Water Sci Technol., 37, 135e142 (1998) 15 Magrí, A., Corominas, L L., López, H., Campos, E., Balaguer, M., Colprim, J., and Flotats, X.: A model for the simulation of the SHARON process: pH as a key factor, Environ Technol., 28, 255e265 (2007) 16 Anthonisen, A C., Loehr, R C., Prakasam, T B S., and Srinath, E G.: Inhibition of nitrification by ammonia and nitrous acid, J Water Pollut Cont Fed., 40, 835e852 (1976) 17 American Public Health Association: Standard methods for the examination of water and wastewater, 20th ed United Book Press, Washington DC, USA (1998) 18 Ministry of Natural Resources and Environment National Technical Regulation on Wastewater of the Solid Waste Landfill Sites: TCVN 6638:2000 water quality e determination of nitrogen e catalytic digestion after reduction with devarda’s alloy, Vietnam Regulation No 25:2009/BTNMT (2009) 19 Fujii, T., Sugino, H., Rouse, J., and Furukawa, K.: Characterization of the microbial community in an anaerobic ammonium-oxidizing biofilm cultured on a nonwoven biomass carrier, J Biosci Bioeng., 94, 412e418 (2002) 20 Qiao, S., Nishiyama, T., Fuhii, T., Bhatti, Z., and Furukawa, K.: Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process, J Biodegrad., 23, 157e164 (2012) 21 Young-Ho, Ahn: Sustainable nitrogen elimination biotechnologies: a review, Process Biochem., 41, 1709e1721 (2006) 22 Strous, M., Kuenen, J G., and Jetten, M.: Key physiological of anaerobic ammonium oxidation, Appl Microbiol Biotechnol., 65, 3248e3250 (1999) 23 Khin, T and Annachhatre, A P.: Novel microbial nitrogen removal processes, Biotechnol Adv., 22, 519e532 (2004) Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 VO AND NGUYEN 24 Ganigue, R., Gabarró, J., López, H., Ruscalleda, M., Balaguer, M D., and Colprim, J.: Combining partial nitritation and heterotrophic denitritation for the treatment of landfill leachate previous to an anammox reactor, Water Sci Technol., 61, 1949e1955 (2010) 25 Wang, C C., Lee, P H., Kumar, M., Huang, Y T., Sung, S., and Lin, J G.: Simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale landfill leachate treatment plant, J Hazard Mater., 175, 622e628 (2010) 26 Yamagiwa, Y and Furukawa, K.: Single-Stage nitrogen removal using anammox and partial nitritation (SNAP) with acrylic pile fabrics as biomass carries, in: The First International Anammox Symposium Conference Proceeding, Kumamoto University, Japan, 217e224 (2011) 27 Egli, K., Fanger, U., Alvarez, P J J., Siegrist, H., van der Meer, J R., and Zehnder, A J B.: Enrichment and characterization of an anammox bacterium J BIOSCI BIOENG., from a rotating biological contactor treating ammonium-rich leachate, Arch Microbiol., 175, 198e207 (2001) 28 Furukawa, K., Rouse, J D., Imajo, U., Nakamura, K., and Ishida, H.: Anaerobic oxidation of ammonium confirmed in continuous flow treatment using a nonwoven biomass carrier, Jpn J Water Treat Biol., 38, 87e94 (2002) 29 Schmid, M., Twachtmann, U., Klein, M., Strous, M., Juretschko, S., Jetten, M., Metzger, J W., Schleifer, K H., and Wagner, M.: Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation, Syst Appl Microbiol., 23, 93e106 (2000) 30 Biec, H N.: The research of partial nitritation process using SBR technology to treat old landfill leachate, Master’s graduate thesis e Ho Chi Minh City University of Technology, Vietnam (2013) Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier, J Biosci Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.01.017 ... simulating pretreated leachate was used as the influent for the start-up phase for the SNAP sludge Then, the synthetic leachate and old leachate were mixed together at different ratios (the ratios... of biomass carriers and biomass carrier fixed on steel frames Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using. .. (8) and Hien et al (10) because the SNAP reactor was operated under a high loading rate using real landfill leachate as influent and used real landfill leachate Therefore, a higher DO supply was