当前位置： >>
污水处理厂 外文文献
Appl Microbiol Biotechnol DOI 10.-013-4744-xENVIRONMENTAL BIOTECHNOLOGYOne-stage partial nitritation/anammox at 15 °C on pretreated sewage: feasibility demonstration at lab-scaleHaydée De Clippeleir & Siegfried E. Vlaeminck & Fabian De Wilde & Katrien Daeninck & Mariela Mosquera & Pascal Boeckx & Willy Verstraete & Nico BoonReceived: 26 November 2012 / Revised: 28 January 2013 / Accepted: 30 January 2013 # Springer-Verlag Berlin Heidelberg 2013Abstract Energy-positive sewage treatment can be achieved by implementation of oxygen-limited autotrophic nitrification/denitrification (OLAND) in the main water line, as the latter does not require organic carbon and therefore allows maximum energy recovery through anaerobic digestion of organics. To test the feasibility of mainstream OLAND, the effect of a gradual temperature decrease from 29 to 15 °C and a chemical oxygen demand (COD)/N increase from 0 to 2 was tested in an OLAND rotating biological contactor operating at 55C60 mg NH4+CNL?1 and a hydraulic retention time of 1 h. Moreover, the effect of the operational conditions and feeding strategies on the reactor cycle balances, including NO and N2O emissions were studied in detail. This study showed for the first time that total nitrogen removal rates of 0.5 g NL?1 day?1 can be maintained when decreasing the temperature from 29 to 15 °C and when low nitrogen concentration and moderate COD levels are treated. Nitrite accumulation together with elevated NO and N2O emissions (5 % of N load) were needed to favor anammox compared with nitratation at low free ammonia (&0.25 mg NL?1), low free nitrous acid (&0.9 μg NL?1), andElectronic supplementary material The online version of this article (doi:10.-013-4744-x) contains supplementary material, which is available to authorized users. H. De Clippeleir : S. E. Vlaeminck : F. De Wilde : K. Daeninck : M. Mosquera : W. Verstraete : N. Boon (*) Laboratory for Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Gent, Belgium e-mail: Nico.Boon@UGent.be P. Boeckx Laboratory of Applied Physical Chemistry (ISOFYS), Ghent University, Coupure Links 653, 9000 Gent, Belgiumhigher DO levels (3C4 mg O2 L?1). Although the total nitrogen removal rates showed potential, the accumulation of nitrite and nitrate resulted in lower nitrogen removal efficiencies (around 40 %), which should be improved in the future. Moreover, a balance should be found in the future between the increased NO and N2O emissions and a decreased energy consumption to justify OLAND mainstream treatment. Keywords Energy self-sufficient . Nitrospira . Nitric oxide . Nitrous oxide . DeammonificationIntroduction Currently, around 40 full-scale one-stage partial nitritation/anammox plants are implemented to treat highly loaded nitrogen streams devoid in carbon (Vlaeminck et al. 2012). This process, known under the acronyms oxygen-limited autotrophic nitrification/denitrification (OLAND) (Kuai and Verstraete 1998), deammonification (Wett 2006), completely autotrophic nitrogen removal over nitrite (Third et al. 2001), etc., showed highly efficient and stable performance when treating digestates from sewage sludge treatment plants and industrial wastewaters (Wett 2006; Abma et al. 2010; Jeanningros et al. 2010). For clarity, one-stage partial nitritiation/anammox processes will be referred to as OLAND in this work. From an energy point of view, the implementation of the OLAND process for the treatment of sewage sludge digestate decreased the net energy consumption of a municipal wastewater treatment plant (WWTP) by 50 %, with a combination of a lower aeration cost in the side stream and the opportunity to recover more organics from the mainstream (Siegrist et al. 2008). Moreover, when co-digestion of kitchen waste was applied, an energy neutralAppl Microbiol BiotechnolWWTP was achieved (Wett et al. 2007). To fully recover the potential energy present in wastewater, a first idea of a new sustainable wastewater treatment concept was reported (Jetten et al. 1997). Recently, a “ZeroWasteWater” concept was proposed which replaces the conventional activated sludge system by a highly loaded activated sludge step (A-step), bringing as much as organic carbon (chemical oxygen demand (COD)) as possible to the solid fraction, and a second biological step (B-step) removing the residual nitrogen and COD with a minimal energy demand (Verstraete and Vlaeminck 2011). Subsequently, energy is recovered via anaerobic digestion of the primary and secondary sludge. For the B-step in the main line, OLAND would potentially be the best choice as this process can work at a low COD/N ratio, allowing maximum recovery of COD in the A-step. Moreover, it was calculated that if OLAND is implemented in the main water treatment line and a maximum COD recovery takes place in the A-step, a net energy gain of the WWTP of 10 Wh inhabitant equivalent (IE)?1 day?1 is feasible (De Clippeleir et al. 2013). To allow this energy-positive sewage treatment, OLAND has to face some challenges compared with the treatment of highly loaded nitrogen streams (&250 mg NL?1). A first difference is the lower nitrogen concentration to be removed by OLAND. Domestic wastewater after advanced concentration will still contain around 30C100 mg NL?1 and 113C 300 mg CODL?1 (Metcalf and Eddy 2003; Tchobanoglous et al. 2003; Henze et al. 2008). High nitrogen conversion rates (around 400 mg NL?1 day?1) by the OLAND process can be obtained at nitrogen concentrations of 30C60 mg N L?1 and at low hydraulic retention times (HRT) of 1C2 h (De Clippeleir et al. 2011). A second challenge is the low temperature at which OLAND should be operated (10C15 °C compared with 34 °C). Several studies already described the effect of temperature on the activity of the separate microbial groups (Dosta et al. 2008; Guo et al. 2010; Hendrickx et al. 2012). Only a few studies showed the long-term effect of a temperature decrease below 20 °C on the microbial balances of anoxic and aerobic ammonium-oxidizing bacteria (AnAOB and AerAOB) and nitrite-oxidizing bacteria (NOB) at nitrogen concentrations above 100 mg N L?1 (Vazquez-Padin et al. 2011; Winkler et al. 2011). However, the combination of low temperature and low nitrogen concentration was never tested on a co-culture of AerAOB, AnAOB, and NOB before. At temperatures around 15 °C, maintaining the balance between NOB and AnAOB and the balance between NOB and AerAOB will get more challenging since the growth rate of NOB will become higher than the growth rate of AerAOB (Hellinga et al. 1998). Therefore, it will not be possible to wash out NOB based on overall or even selective sludge retention. The third and main challenge in this application will therefore be the suppression of NOB at temperature ranges of 10C20 °Cand at nitrogen concentration ranges of 30C60 mg NL?1 (low free ammonia and low nitrous acid), which was not shown before. A final fourth challenge will include the higher input of organics at moderate levels of 90C240 mg biodegradable CODL?1 in the wastewater. Depending on the raw sewage strength, COD/N ratios between 2 and 3 are expected after the concentration step, which is on the edge of the described limit for successful OLAND (Lackner et al. 2008). The presence of organics could result in an extra competition of heterotrophic denitrifiers with AerAOB for oxygen or with AnAOB for nitrite or organics, since certain AnAOB can denitrify consuming organic acids (Kartal et al. 2007). In this study, the challenges 2 to 4, were evaluated in an OLAND rotating biological contactor (RBC). This reactor at 29 °C was gradually adapted over 24, 22, and 17 to 15 °C under synthetic wastewater conditions (60 mg N L ?1 , COD/N of 0). Additionally, the COD/N ratio of the influent was increased to 2 by supplementing NH4+ to diluted sewage to simulate pretreated sewage. The effect of the operational conditions and feeding strategies on the reactor cycle balances, including gas emissions and microbial activities were studied in detail. An alternative strategy to inhibit NOB activity and as a consequence increase AnAOB activity at low temperatures based on NO production was proposed.Materials and methods OLAND RBC The lab-scale RBC described by De Clippeleir et al. (2011) was further optimized at 29 °C by an increase in the influent nitrogen concentration from 30 to 60 mg N L?1 and a limitation of the oxygen input through the atmosphere by covering the reactor before this test was started. The reactor was based on an air washer LW14 (Venta, Weingarten, Germany) with a rotor consisting of 40 discs interspaced at 3 mm, resulting in a disc contact surface of 1.32 m2. The reactor had a liquid volume of 2.5 L, immersing the discs for 55 %. The latter was varied over the time of the experiment. The reactor was placed in a temperature-controlled room. The DO concentration was not directly controlled. In this work, continuous rotation was applied at a constant rotation speed of 3 rpm, which allowed mixing of the water phase. RBC operation The RBC was fed with synthetic wastewater during phases I to VII. From phase VIII onwards, the COD/N was gradually increased (phases VIIICX) to 2 (phases XICXIII). The synthetic influent of an OLAND RBC, consisted of (NH4)2SO4 (55C60 mg NL?1), NaHCO3 (16 mg NaHCO3 mg?1 N), andAppl Microbiol BiotechnolKH2PO4 (10 mg PL?1). Pretreated sewage was simulated by diluting raw sewage of the communal WWTP of Gent, Belgium (Aquafin). The raw wastewater after storage at 4 °C and settlement contained 23C46 mg NH4+CN L?1, 0.2C0.4 mg NO2?CNL?1, 0.4C2.7 mg NO3?CNL?1, 23C46 mg KjeldahlCNL?1, 3.8C3.9 mg PO43?CPL-1, 26C27 mg SO42?CS L?1, 141C303 mg CODtot L?1, and 74C145 mg CODsol L?1. The raw sewage was diluted by factors 2C3 to obtain COD values around 110 mg CODtot L?1 and by addition of (NH4)2SO4 to obtain final COD/N values around 2. The reactor was fed in a semi-continuous mode: two periods of around 10 min/h for phases ICXI and one period of 20 min/h for phases XII and XIII. The influent flow range varied from 47 to 65 Lday?1 and the reactor volume from 3.7 to 2.5 L (during 78 and 55 % submersion, respectively). Corresponding HRT are displayed in Tables 1 and 2. Reactor pH, DO, and temperature were daily monitored and influent and effluent samples were taken at least thrice a week for ammonium, nitrite, nitrate, and COD analyses. Detection of AerAOB, NOB, and AnAOB with FISH and qPCR For NOB and AnAOB, a first genus screening among the most commonly present organisms was performed by fluorescent in-situ hybridization (FISH) on biomass of days 1 (high temperature) and 435 (low temperature and COD presence). A paraformaldehyde (4 %) solution was used for biofilm fixation, and FISH was performed according to Amann et al. (1990). The Sca1309 and Amx820 probeswere used for the detection of Cand. Scalindua and Cand. Kuenenia & Brocadia, respectively, and the NIT3 and Ntspa662 probes and their competitors for Nitrobacter and Nitrospira, respectively (Loy et al. 2003). This showed the absence of Nitrobacter and Scalindua (Table S1 in the Electronic supplementary material (ESM)). Biomass samples (approximately 5 g) for nucleic acid analysis were taken from the OLAND RBC at days 1, 60, 174, 202, 306, 385, 399, and 413 of the operation. DNA was extracted using FastDNA? SPIN Kit for Soil (MP Biomedicals, LLC), according to the manufacturer’s instructions. The obtained DNA was purified with the Wizard? DNA Clean-up System (Promega, USA) and its final concentration was measured spectrophotometrically using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies). The SYBR Green assay (Power SyBr Green, Applied Biosystems) was used to quantify the 16S rRNA of AnAOB and Nitrospira sp. and the functional amoA gene for AerAOB. The primers for quantitative polymerase chain reactions (qPCR) for detection of AerAOB, NOB, and AnAOB were amoA-1FCamoA-2R (Rotthauwe et al. 1997), NSR1113fCNSR1264r (Dionisi et al. 2002), and Amx818fCAmx1066r (Tsushima et al. 2007), respectively. For bacterial amoA gene, PCR conditions were: 40 cycles of 94 °C for 1 min, 55 °C for 1 min, and 60 °C for 2 min. For the amplification of Nitrospira sp. 16S rRNA gene, 40 cycles of 95 °C for 1 min, 50 °C for 1 min, and 60 °C for 1 min were used while for AnAOB 16S rRNA the PCR temperature program was performed by 40 cycles of 15 s at 94 °C and 1 min at 60 °C. Plasmid DNAs carrying NitrospiraTable 1 Effect of temperature decrease on the operational conditions and performance of OLAND RBC reactor Phase Period (days) Immersion level (%) Temperature (°C) Operational conditions DO (mg O2 L?1) pH (?) HRT (h) FA (mg NL?1) FNA (μg NL?1) Performance Total N removal efficiency (%) Relative NO3? prod (% of NH4+ consa) Relative NO2? accum (% of NH4+ cons) AerAOB activity (mg NH4+CNL?1 day?1) NOB activity (mg NO2CNL?1 day?1) AnAOB activity (mg Ntot L?1 day?1) I 1C21 78 29±2 1.1±0.2 7.5±0.1 1.85±0.04 0.35±0.18 0.3±0.1 54±5 7±1 2±4 267±38 0±0 412±38 II 22C35 78 24±1 1.3±0.2 7.5±0.1 1.84±0.09 0.36±0.18 0.3±0.2 52±5 7±1 3±4 267±49 0±0 403±37 III 36C61 78 22±0.6 1.4±0.1 7.5±0.1 1.73±0.04 0.34±0.14 0.4±0.2 49±9 7±1 5±5 260±52 0±0 368±76 IV 62C210 78 17±1.2 1.7±0.3 7.6±0.1 1.86±0.11 0.36±0.13 0.4±0.1 34±9 14±6 15±5 260±53 9±12 248±67 V 210C263 55 16±0.9 2.8±0.4 7.7±0.1 1.09±0.02 0.25±0.16 0.9±0.4 36±9 18±9 30±8 811±229 60±94 448±117 VI 263C274 78 15±0.8 2.4±0.2 7.7±0.1 1.57±0.02 0.33±0.17 0.6±0.1 36±9 16±3 26±6 460±44 20±5 305±74 VII 275C306 55 14±0.4 3.1±0.2 7.8±0.1 1.09±0.02 0.13±0.04 0.9±0.2 42±4 21±4 31±5 986±71 85±25 529±75DO dissolved oxygen, HRT hydraulic retention time, FA free ammonia, FNA free nitrous acid, cons consumption, prod production, accum accumulation, tot totalaNH4+ consumption is corrected for nitrite accumulationAppl Microbiol Biotechnol Table 2 Effect of COD/N increase on the operational conditions and performance of OLAND RBC reactor Phase Period (days) Immersion level (%) COD/N (-) Feeding regime (pulsesh?1) Operational conditions DO (mg O2 L?1) pH (?) HRT (h) FA (mg NL?1) FNA (μg NL?1) Performance Total N removal efficiency (%) Relative NO3? prod (% of NH4+ consa) Relative NO2? accum (% of NH4+ cons) AerAOB activity (mg NH4+CNL?1 day?1) NOB activity (mg NO2?CNL?1 day?1) AnAOB activity (mg Ntot L?1 day?1) COD removal rates were negligible in all phases DO dissolved oxygen, HRT hydraulic retention time, FA free ammonia, FNA free nitrous acid, cons consumption, prod production, accum accumulation, tot totalaVIII 355C361 55 0.5 2 2.9±0.3 7.8±0.02 1.06±0.11 0.10±0.05 0.4±0.1 36±5 42±5 20±4 592±15 257±19 385±86IX 362C369 55 1 2 2.5±0.6 7.7±0.1 1.03±0.02 0.04±0.05 0.2±0.2 45±18 43±12 10±10 446±31 294±81 452±205X 370C374 55 1.5 2 2.4±0.3 7.6±0.02 0.92±0.02 0.15±0.05 0.2±0.01 23±3 63±2 5±1 238±28 465±60 262±39XI 375C406 55 2 2 3.0±0.7 7.6±0.1 0.94±0.05 0.21±0.10 0.3±0.1 28±6 50±6 8±3 352±73 352±84 355±73XII 407C421 55 2 1 3.6±0.3 7.6±0.2 1.10±0.05 0.23±0.12 0.2±0.1 23±13 62±18 7±4 289±138 427±115 281±159XIII 422C435 55 2 1 3.2±0.3 7.6±0.1 1.06±0.2 0.04±0.02 0.6±0.2 42±3 46±6 13±6 600±204 394±76 481±73NH4+ consumption is corrected for nitrite accumulationand AnAOB 16S rRNA gene and AerAOB functional AmoA gene, respectively, were used as standards for qPCR. All the amplification reactions had a high correlation coefficient (R2 &0.98) and slopes between ?3.0 and ?3.3. Detailed reactor cycle balances For the measurements of the total nitrogen balance, including the NO and N2O emissions, the OLAND RBC was placed in a vessel (34 L) which had a small opening at the top (5 cm2). In this vessel, a constant upward air flow (around 1 ms?1 or 0.5 L s-1) was generated to allow calculations of emission rates. On the top of the vessel (air outlet), the NO and N2O concentration was measured, off- and online, respectively. NH3 emissions were negligible in a RBC operated at about 2 mg NH3C NL?1 (Pynaert et al. 2003). Since FA levels in the current study are about ten times lower, NH3 emissions were not included. In the water phase, ammonium, nitrite, nitrate, hydroxylamine (NH2OH), N2O, and COD concentrations were measured. Moreover, DO concentration and pH values were monitored. The air flow was measured with Testo 425 hand probe (Testo, Ternat, Belgium). Chemical analyses Ammonium (Nessler method) was determined according to standard methods (Greenberg et al. 1992). Nitrite and nitratewere determined on a 761 compact ion chromatograph equipped with a conductivity detector (Metrohm, Zofingen, Switzerland). Hydroxylamine was measured spectrophotometrically (Frear and Burrell 1955). The COD was determined with NANOCOLOR? COD 1500 en NANOCOLOR? COD 160 kits (Macherey-Nagel, Düren, Germany). The volumetric nitrogen conversion rates by AerAOB, NOB, and AnAOB were calculated based on the measured influent and effluent compositions and the described stoichiometries, underestimating the activity of AnAOB by assuming that all COD removed was anoxically converted with nitrate to nitrogen gas (Vlaeminck et al. 2012). DO and pH were measured with respectively, a HQ30d DO meter (Hach Lange, Düsseldorf, Germany) and an electrode installed on a C833 meter (Consort, Turnhout, Belgium). Gaseous N2O concentrations were measured online at a time interval of 3 min with a photo-acoustic infrared multi-gas monitor (Brüel & Kj?r, Model 1302, N?rem, Denmark). Gas grab samples were taken during the detailed cycle balance tests for NO detection using Eco Physics CLD 77AM (Eco Physics AG, Duernten, Switzerland), which is based on the principle of chemiluminescence. For dissolved N2O measurements, a 1mL filtered (0.45 μm) sample was brought into a 7-mL vacutainer (?900 hPa) and measured afterwards by pressure adjustment with He and immediate injection at 21 °C in a gas chromatograph equipped with an electron capture detector (Shimadzu GC-14B, Japan).Appl Microbiol BiotechnolResults Effect of temperature decrease During the reference period (29 °C), a well-balanced OLAND performance (Fig. 1; Table 1) was reached with minimal nitrite accumulation (2 %) and minimal nitrate production (7 %). This was reflected in an AerAOB/AnAOB activity ratio of 0.6 (Table 1, phase I). The total nitrogen removal rate was on average 470 mg N L ?1 day ?1 or 1314 mg Nm?2 day?1, and the total nitrogen removal efficiency was 54 %. Decreasing the temperature from 29 to 24 °C and further to 22 °C over the following 40 days, did not result in anyFig. 1 Phases ICVII: effect of temperature decrease on the volumetric rates (top) and nitrogen concentrations (bottom)significant changes of the operational conditions (Table 1; phases ICIII), performance of the reactor (Fig. 1) or abundance of the bacterial groups (qPCR; Fig. S1 in the ESM). However at 17 °C, a decrease in total nitrogen removal efficiency was observed (Table 1; phase IV). An imbalance between the AerAOB and the AnAOB was apparent from a stable AerAOB activity yet a declining AnAOB activity. Moreover, NOB activity was for the first time detected in spite of free ammonia (FA) and free nitrous acid (FNA) concentrations did not change (Table 1; phase IV). Moreover, no significant differences in abundance of NOB, AerAOB, and AnAOB could be detected with qPCR (Fig. S1 in the ESM). However, DO concentrations started to increase during that period from 1.4 to 1.7 mg O2 L?1. As the availability ofAppl Microbiol Biotechnoloxygen through the liquid phase did not seem to be satisfactory to counteract the decrease in ammonium removal efficiency, the immersion level was lowered to 55 % to increase the availability of oxygen through more air-biofilm contact surface. Consequently, the volumetric loading rate increased (factor 1.7) due to the decrease in reactor volume (day 210, Fig. 1). This action allowed higher ammonium removal efficiencies due to higher AerAOB activities (factor 3). AnAOB activity increased with a similar factor as the volumetric loading rate (1.8 compared with 1.7) consequently resulting in an increased imbalance between these two groups of bacteria (Table 1; phase V). Moreover, although the FNA increased with a factor 2, the NOB activity increased with a factor 7, resulting in a relative nitrate production of 30 % (Table 1; phase V). As NOB activity prevented good total nitrogen removal efficiencies, the immersion level was increased again to 78 % (day 263; Fig. 1). This resulted indeed in a lower NOB activity (Table 1; phase VI). However, also the AerAOB activity decreased with the same factor, due to the lower availability of atmospheric oxygen. Therefore, the reactor was subsequently operated again at the lower immersion level (55 %) to allow sufficient aerobic ammonium conversion. The latter allowed a stable removal efficiency of 42 %. The AnAOB activity gradually increased to a stable anoxic ammonium conversion rate of 529 mg NL?1 day?1. During the synthetic phase, no changes in AerAOB, AnAOB, and NOB abundance were measured with qPCR (Fig. S1 in the ESM). The effluent quality was however not optimal as still high nitrite (around 15 mg NL?1) and nitrate (around 13 mg NL?1) levels were detected. Effect of COD/N increase The synthetic feed was gradually changed into pretreated sewage by diluting raw sewage and adding additional nitrogen to obtain a certain COD/N ratio. During the first 3 weeks of this period (Fig. 2), the COD/N ratio was gradually increased from 0.5 to 2. Due to the short adaptation periods (1 week per COD/N regime), the performance was unstable (Fig. 2; Table 2, phases VIIICXI). Compared with the end of the synthetic period (phase VII), operation at a COD/N ratio of 2 (phase XI) resulted in a sharp decrease in nitrite accumulation (Fig. 2) and an increase in the ammonium and nitrate levels. This indicated increased NOB activity (factor 4), decreased AerAOB (factor 3) and decreased AnAOB (factor 2) activity (Tables 1 and 2). To allow higher nitrogen removal rates, the HRT was increased from 0.94 to 1.1 h, by decreasing the influent flow rate. Moreover, the feeding regime was changed from two pulses of 10 min in 1 h to one period of 20 min/h. These actions did not significantly decrease the effluent nitrogen concentration (Fig. 2) and did not influence the microbial activities (Table 2, phase XII). Therefore the loading rate was again increased to the levelsbefore phase XII. However, the single-pulse feeding was maintained. This resulted in high ammonium removal efficiencies and therefore low ammonium effluent concentration around dischargeable level (4 ± 1 mg NH4+CN L?1; Fig. 2). Nitrate and nitrite accumulation were not counteracted by denitrification as only 0.02 mg CODL?1 day?1 was removed. Therefore, nitrite and nitrate levels were still too high to allow effluent discharge. The total nitrogen removal efficiency (42 %) and rate (549±83 mg NL?1 day?1 or 1,098± 106 mg N m?2 day?1) at COD/N ratios of 2 was similar as during the synthetic period (phase VII). Compared with the reference period at 29 °C, the total nitrogen removal rate did not changed significantly (470±43 versus 549±83 mg NL?1 day?1 at high and low temperatures, respectively). The 22 % lowered removal efficiency was merely due to an increased nitrogen loading rate. Nitratation and NO/N2O emissions At the end of the synthetic phase (phase VII) and the end of the experiment (phase XIII), the total nitrogen balance of the reactor was measured. A total nitrogen balance was obtained by measuring all nitrogen species (NH4+, NO2?, NO3?, NH2OH, and N2O) in the liquid phase and N2O and NO in the gas phase. A constant air flow, diluting the emitted N2O and NO concentrations was created over the reactor to measure gas fluxes over time. The effect of the loading rate, feeding pattern, and concentration of nitrite and ammonium on the total nitrogen balance in the reactor were tested (Table 3). NH2OH measurements showed low concentrations (&0.2 mg NL?1) in all tests, making it difficult to link the profiles with the N2O emission. Lowering the loading rate by increasing the HRT (Table 3, test B) increased the DO values and allowed higher DO fluctuations over time at synthetic conditions. Moreover, NOB activity increased significantly resulting in lower total nitrogen removal efficiencies and high levels of nitrate in the effluent (Table 3, test B). The relative N2O emissions did not change and were relatively high (6 % of N load). However, the concentration of N2O in the liquid and in the gas phase decreased with a factor 2 (Table 3). When pretreated sewage was fed to the reactor, the OLAND RBC was operated at lower nitrite concentration, while similar ammonium and nitrate concentrations were obtained (Table 3, test C). The lower nitrite concentrations however did not result in lower N2O emission rates. When the feeding regime was changed to a more continuous-like operation (4 pulses/h), the N2O emission increased significantly, while NO emission remained constant (Table 3, test D). Due to the lower ammonium removal efficiency (65 compared with 81 %), but similar relative nitrite and nitrate accumulation rate, the total nitrogen removal efficiency decreased.Appl Microbiol Biotechnol Fig. 2 Phases VIIICXIII: effect of COD/N increase on the volumetric rates (top) and nitrogen concentrations (bottom). Data during the N balance tests (days 424C431) were not incorporated in the figure but are shown in Table 3When a nitrite pulse was added just after feeding, about 20 mg NO2?CNL?1 was obtained in the reactor. This did increase the NO and N2O emissions significantly (p&0.05) compared with the same feeding pattern (Table 3, tests CCE). Although similar constant total nitrogen removal efficiencies were obtained during this operation, a significant (p&0.05) decrease in the relative nitrate production was observed. The latter was mainly caused by a global increase in AnAOB activity. In the last test (F), the influent ammonium concentration was doubled, leading to higher ammonium and also FA concentrations (1 ± 0.4 mg N L ?1 compared with 0.1±0.4 mg NL?1). Due to overloading of the system, the total nitrogen removal efficiency decreased. However, at these conditions a lower relative nitrate pro due to a decrease in NOB and increase in AnAOB activity (Table 3, test F). Together withthis, increased NO and N2O emissions were observed. As the influence of the nitrogen loading and DO concentration could be considered minor in this test range (Fig. S2 in the ESM), these tests show a relation between increased NO emissions and decreased relative nitrate productions (Table 3). When the activity during the feeding cycle was studied in more detail, it could be concluded that the highest nitrogen conversion rates took place during the feeding period, which was characterized by a high substrate availability and high turbulence (Fig. 3). As the HRT is only 1 h, the reactor volume is exchanged in 20 min. During this phase, ammonium increased, while nitrite and nitrate concentrations decreased due to dilution (Figs. S3, S4, and S5 in the ESM). The NOB/AnAOB ratio was around 1, which means that NOB were able to take twice as much nitrite than AnAOBAppl Microbiol Biotechnol Table 3 Operational parameters and nitrogen conversion rates during the six different RBC operations which differ from feeding composition and feeding regime (volume at 2.5 L and 50 % immersion of the discs, days 307C309 for synthetic feed, and days 424C431) Reactor phase Test Additive Feeding regime (pulses/h) Total N loading rate (mg NL?1 day?1) Temperature water (°C) DO (mg O2 L?1) pH (-) Ammonium out (mg NL?1) Nitrite out (mg NL?1) Nitrate out (mg NL?1) NH4+ oxidation rate (mg NL?1 day?1) Relative nitrite accumulation (%) Relative nitrate production (%) Total efficiency (%) AerAOB activity (mg NH4+CNL?1 day?1) NOB activity (mg NO2?CNL?1 day?1) AnAOB activity (mg Ntot L?1 day?1) N2O in liquid (μg NL?1) NO emission (mg Nday?1) N2O emission (mg Nday?1) % N2O emission on loadingaVII (synthetic) Aa C 2 1,169 15±0.3 2.9±0.1 7.6±0.06 9±1 14±2 17±3 895±22 25±3 36±8 38±4 658±88 174±59 205±38 64±46 0.53±0.03 151±28 5.1±1.0 B C 2 585 16±0.2* 3.7±0.6* 7.6±0.05 1.4±1* 13±1 37±6* 509±2* 20±1* 76 ±6* 17±4* 469±17* 299±28* 49±13* 30±22* n.d. 93±23* 6.4±1.6*XIII (pretreated sewage) Ca C 1 1,340 14±0.4 4.0±0.1 7.6±0.04 11±3 6±1 18±2 1,051±73 14±3 48±1 35±3 827±44 375±38 234±20 78±12 0.66±0.06 170±19 5.0±0.6 D C 4 1,554 15±0.1* 3.2±0.1* 7.6±0.01 19±3* 6±0.4 16±1* 957±89 15±1 47±3 28±4* 781±57 342±24* 218±29 104±29* 0.74±0.08 179±6* 4.5±0.2* ENO2? 1 1,737 16±0.1* 3.3±0.1* 7.6±0.02 12±1 18±2* 18±0.4 1,053±16 8±4* 42±2* 32±2 795±30 362±13 263±15* 61±13 1.65±0.18* 274±37* 6.2±0.8* F NH4+ 1 2,718 15±0.4 3.2±0.1* 7.8±0.02* 58±4* 9±0.3* 20±0.4 1,285±93* 15±1 34±3* 27±4* 938±46* 277±18* 354±49* 74±4 0.82±0.1* 202±18* 3.0±0.3*Reference period for synthetic and pretreated sewage*p&0.05, significant differences compared with reference period Fig. 3 Detailed NO/N2O monitoring during the reference test (Table 3, test C) and when nitrite was pulsed (Table 3, test E) and effect on AerAOB, AnAOB, and NOB activity during the different phases of the feeding cycle. Significant differences in AerAOB, AnAOB, NOB, and NO/N2O concentration compared with the reference period are indicated with asterisks, circles, double quotation mark, and plus sign, respectivelyAppl Microbiol Biotechnoldid, as the latter also consumed ammonium (Fig. 3). After the feeding period, a lag phase of the ammonium increase was observed, because the reactor liquid was not homogenously mixed yet. After mixing (10 min after feeding) was established, a N2O peak was reached during every test (Figs. S3, S4, and S5 in the ESM). At this point, during the reference period with pretreated sewage (test C) total activity decreased and a very low NOB activity was observed (Fig. 3). Moreover, the NOB/AnAOB ratio decreased to 0.4 (Fig. 3, test C), which means that during these conditions nitrite consumption by AnAOB was higher than nitrite consumption by NOB. The increased relative AnAOB activity was more pronounced when a higher NO and N2O peak were present (test E). The latter was caused by an increased nitrite concentration in the reactor. When N2O concentration started to decrease again (last 20 min of feeding regime), nitrite consumption by NOB was again higher than the nitrite consumption by AnAOB (Fig. 3).Discussion Effect of temperature decrease Average temperatures of sewage in west European region are around 17 °C, with a minimum of 8 °C and a maximum of 29 °C (Mollen, personal communication). Therefore, the temperature of the OLAND RBC was decreased from 29 to 15 °C. In contrast to the optimal microbial balance at temperatures of &20 °C, excess nitrite and nitrate formation was observed at lower temperatures. Improved operational conditions (O2 availability) resulted in similar nitrogen conversion rates for AerAOB and AnAOB at lower temperature (&20 °C) compared with the reference period at 29 °C. The gradual adaptation of AerAOB and AnAOB to low temperatures was a key to maintaining high activities, since activities deteriorated more with sudden temperature drops (Dosta et al. 2008; Guo et al. 2010). Similar long-term effects of temperature on AerAOB activity (Guo et al. 2010) and AnAOB activity (Hu et al. 2011; Hendrickx et al. 2012) were observed before. Due to the higher DO concentration at lower temperatures and the higher exposure to atmospheric oxygen at lower immersion levels, the oxygen penetration depth possibly increased and zones constantly submersed before were exposed to oxygen, which could not allow an increase in AnAOB activity. On the other hand, higher oxygen inputs were needed at lower temperatures to obtain the same AerAOB activity as at high temperature. The combination of these two factors could have been responsible for the increased nitrite accumulation from phase IV onwards. Therefore, at lower temperature the OLAND performance will be limited by AerAOB activityas their activity guarantees anoxic zones in the biofilm (Vazquez-Padin et al. 2011). Increased AerAOB activities were obtained at high DO levels (three times higher than at 29 °C). This was on one hand caused by a better solubility of oxygen at lower temperatures and on the other hand by a decrease of the immersion level from 78 to 55 %. Although the changes in immersion level, did not always result in a significant DO change (phases IV to V and V to VI), the oxygen availability through contact with the atmosphere was changed drastically. This suggests that oxygen transfer through atmospheric oxygen is more important in this system compared with transfer from dissolved oxygen. Although oxygen concentrations in OLAND systems at high temperature conditions are controlled at levels below 1 mg O2 L?1 to avoid nitrate oxidation by NOB, at low temperatures 2C4 mg O2 L?1 is needed to allow sufficient AerAOB activity (Vazquez-Padin et al. 2011). As nitrite accumulated in the OLAND RBC, oxygen input was probably too high to allow a balanced performance between nitrite production and consumption. Therefore, in practice a bulk DO control system is advisable to obtain a better removal efficiency. Effect of COD/N increase COD addition did not result in a better nitrogen removal efficiency or lower AnAOB activities (Lackner et al. 2008) as almost no COD removal was observed. The 2011 yearround data of sewage in the city of Ghent show a relatively low biodegradable fraction of only 0.57 ± 0.17 at 325 ± 95 mg COD/L. It is likely that a considerable part of the biodegradable COD degraded during storage or in the influent vessel prior to feeding the OLAND RBC. In practice, the use of fresh sewage with a higher biodegradable fraction is expected to have more impact. The reason for the change in performance despite the low biodegradable COD level was not clear and was possibly due to adaptation of the microbial community to a new ‘background’ matrix. Nevertheless, it has been already successfully demonstrated that anammox can co-exist with heterotrophic denitrifiers at COD/N ratios of 2.2 (Desloover et al. 2011). Therefore, it should be possible to obtain high nitrogen removal efficiencies without the loss of AnAOB activities at mainstream conditions. NOB-AnAOB competition at mainstream conditions Although Nitrospira sp. were present from days 0 to 375 (phase ICX) at a stable level of around 40 copiesng?1 DNA, at temperatures below 20 °C (day 61, phase IV) NOB activity increased significantly (Fig. S1 in the ESM). Although NOB activity increased, nitrite accumulated in the system due to a higher increase in AerAOB rates (Tables 1 and 2). Moreover, when COD was added andAppl Microbiol Biotechnollower nitrogen concentrations (55 mg N L?1 instead of 60 mg NL?1) were fed (phases X to XIII), relative nitrate productions up to 62 % were observed (Table 2, phase XII). FA and FNA concentrations (Tables 1 and 2) were in all phases too low to suppress nitratation (Anthonisen et al. 1976). Moreover, oxygen inputs (mainly through atmospheric contact) were rather high to allow sufficient nitritation, which could also have stimulated NOB growth and activity. Therefore, for mainstream treatment other strategies beside FA, FNA and oxygen limitation should be applied to suppress nitratation. Detailed nitrogen balance tests showed that the different feeding strategies did not affect the microbial balance in the system (Table 3). However, pulse feeding allowed higher AerAOB activities increasing the ammonium removal efficiency (phase XIII) and continuous-like feeding resulted in a decreased ammonium and as a consequence total nitrogen removal efficiency (Table 3). The increased AerAOB activity at peak loading was probably caused by the transient operation rather than the peak concentration itself (Fig. S4 in the ESM; Table 3). Moreover, a sufficient loading rate was needed to allow a good microbial balance and thus increased AnAOB activity and/or decreased NOB activity (Tables 2, phase XIII and 3, test F). Overloading of the system and therefore obtaining higher FA levels, could inhibit the NOB activity (Table 3, test F) in contrast to the long-term performance at lower FA concentrations. Therefore, the latter could not be responsible for the better microbial balance during reactor operation. However, nitrite accumulation, resulting in higher peaks in NO and N2O production (Table 3) occurred in all well performing periods at low temperature. High NO emissions, initiated by addition of nitrite could increase relative AnAOB activity and decrease NOB activity (Fig. 3). It is well known that NO is toxic to most of the bacteria (Mancinelli and McKay 1983). It has been described before that NO2?-dependent O2 uptake by NOB could reversibly be inhibited by NO at concentrations of 7C448 μg NOCNL?1 (Starkenburg et al. 2008). In contrast, NO is an intermediate for the AnAOB metabolism and high NO concentrations do not affect their activity (Kartal et al. 2010). Therefore, at conditions of high NO concentration, AnAOB can have a competitive advantage compared with NOB. However, from the moment NO decreased below a threshold concentration, NOB activity can increase again (Figs. S4 and S5 in the ESM; Starkenburg et al. (2008)), which was in this study in around 83 % of the cycle time. Although nitritation is stimulated by NO (Zart et al. 2000), it seemed that also AerAOB activity was affected by NO at the NO/N2O peak (Fig. 3). Therefore, a balance should be found between stimulating AnAOB above NOB activity and allowing sufficient nitrite production by AerAOB. The high volumetric loading rate applied, together with the pulse feeding and the nitrite accumulation led to high NO/N2O emissions compared withmesophilic OLAND applications (Kampschreur et al. 2009a; Weissenbacher et al. 2010). This could however be a prerequisite for obtaining low nitratation levels at these mainstream conditions. OLAND application in the main line At 15 °C and a COD/N ratio of 2, high total nitrogen removal rates of 0.5 g NL?1 day?1 or 1 g Nm?2 day?1 were obtained (Fig. 2). However, the total nitrogen removal efficiency was too low to obtain dischargeable effluent (European Commision 1991) and should therefore be optimized in the future by decreasing the nitrite accumulation and/or increasing the denitrification rate. As similar total nitrogen removal rates were obtained at 15 °C compared with 29 °C, the performance was not limited by the mainstream conditions but it was limited by the reactor configuration. Because the discs only had a spacing of 3 mm, regular perforations of the biofilm were needed to allow sufficient diffusion. A better RBC configuration (higher disc distance) or another reactor technology (suspended growth system) could probably allow higher efficiencies due to more efficient diffusion. On the other hand, by a combination of OLAND and conventional nitrification/denitrification in the B-step, a better removal efficiency might be possible. Indeed, the biodegradable fraction of the COD will be higher in practice compared with this study, enabling nitrate removal through denitrification. This can be achieved by only partly replacing the activated sludge by OLAND biomass. To allow AnAOB retention in this system a selectively higher SRT of the OLAND biomass compared with the activated sludge should be maintained. In the case of granular application, the latter can be obtained by implementation of cyclones (Wett et al. 2010), but it should also be possible by inoculation of OLAND biomass on packing material which can be kept in the system by a grid. In this way, OLAND should not be responsible for the total nitrogen removal efficiency of the system and nitrite or nitrate formation can be compensated by denitrification. The latter combination of activated sludge with OLAND can be a potential route for further research. A last point of attention is the high N2O emission (5 % of N load) compared with the high temperature and high nitrogen application, which normally emits around 1 % of its N load as N2 OCN (Kampschreur et al. 2009a; Weissenbacher et al. 2010). Optimization of the operational conditions, especially regarding the nitrite accumulation could probably minimize the emissions (Kampschreur et al. 2009b; Chandran et al. 2011). Therefore, a balance should be found between NOB suppression, N2O emission and energy savings. This study showed for the first time that total nitrogen removal rates of 0.5 g NL?1 day?1 can be maintained when decreasing the temperature from 29 to 15 °C and when lowAppl Microbiol Biotechnolnitrogen concentration and moderate COD levels are treated. Nitrite accumulation together with elevated NO/N2O emissions was needed to allow competition of AnAOB against NOB for nitrite at low FA, low FNA and high DO levels. Although the total nitrogen removal rates were promising, nitrite and nitrate accumulation resulted in lower nitrogen removal efficiencies (around 40 %), which should be improved in the future. Further research should elucidate the mechanism and the level of NO/N2O emission needed to obtain a balanced performance. Moreover, it should be further evaluated if the increased NO/N2O emission can be compensated with a decreased energy consumption to justify OLAND mainstream treatment.Acknowledgments H.D.C. received a Ph.D. grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen, SB-81068), and S.E.V. was supported as a postdoctoral fellow from the Research Foundation Flanders (FWO-Vlaanderen). The authors gratefully thank Aquafin for providing the sewage, Eva Spieck for providing the qPCR standards, and Tom Hennebel, Joachim Desloover, and Simon De Corte for inspiring scientific discussions.ReferencesAbma WR, Driessen W, Haarhuis R, van Loosdrecht MCM (2010) Upgrading of sewage treatment plant by sustainable and costeffective separate treatment of industrial wastewater. Water Sci Technol 61: Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental-studies in microbiology. J Bacteriol 172:762C770 Anthonisen AC, Loehr RC, Prakasam TBS, Srinath EG (1976) Inhibition of nitrification by ammonia and nitrous acid. J Water Pollut Control Fed 48:835C852 Chandran K, Stein LY, Klotz MG, van Loosdrecht MCM (2011) Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems. Biochem Soc Trans 39: De Clippeleir H, Vlaeminck SE, Courtens E, Verstraete W, Boon N (2013) Oxygen-limited autotrophic nitrification/denitrification maximizes net energy gain in technology schemes with anaerobic digestion. In: Renewable energy sources. Academy Publish, Wyoming De Clippeleir H, Yan X, Verstraete W, Vlaeminck SE (2011) OLAND is feasible to treat sewage-like nitrogen concentrations at low hydraulic residence times. Appl Microbiol Biotechnol 90: Desloover J, De Clippeleir H, Boeckx P, Du Laing G, Colsen J, Verstraete W, Vlaeminck SE (2011) Floc-based sequential partial nitritation and anammox at full scale with contrasting N2O emissions. Water Research 45(9): Dionisi HM, Layton AC, Harms G, Gregory IR, Robinson KG, Sayler GS (2002) Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR. Appl Environ Microbiol 68:245C253 Dosta J, Fernandez I, Vazquez-Padin JR, Mosquera-Corral A, Campos JL, Mata-Alvarez J, Mendez R (2008) Short- and long-term effects of temperature on the anammox process. J Hazard Mater 154:688C693European Commision (1991) Council Directive 91/271/EEC of 21 May 1991 concerning urban waste-water treatment. Off J L 135:40C52 Frear DS, Burrell RC (1955) Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal Chem 27: Greenberg AE, Clesceri LS, Eaton AD (1992) Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DC Guo J, Peng Y, Huang H, Wang S, Ge S, Zhang J, Wang Z (2010) Short- and long-term effects of temperature on partial nitrification in a sequencing batch reactor treating domestic wastewater. J Hazard Mater 179:471C479 Hellinga C, Schellen A, Mulder JW, van Loosdrecht MCM, Heijnen JJ (1998) The SHARON process: an innovative method for nitrogen removal from ammonium-rich waste water. Water Sci Technol 37:135C142 Hendrickx TLG, Wang Y, Kampman C, Zeeman G, Temmink H, Buisman CJN (2012) Autotrophic nitrogen removal from low strength waste water at low temperature. Water Res 46: Henze M, Van Loosdrecht M, Ekama GA, Brdjanovic D (2008) Biological wastewater treatment―principles, modelling and design. IWA Publishing, London Hu Z, Lotti T, de Kreuk M, Kleerbezem R, van Loosdrecht M, Kruit J, Jetten M.S.M, Kartal B (2011) Adaptation of anammox bacteria to low temperature in a lab-scale bioreactor. In: 16th European Ncycle Meeting―2nd International Conference on Nitrification (ICoN), Nijmegen, The Netherlands Jeanningros Y, Vlaeminck SE, Kaldate A, Verstraete W, Graveleau L (2010) Fast start-up of a pilot-scale deammonification sequencing batch reactor from an activated sludge inoculum. Water Sci Technol 61: Jetten MSM, Horn SJ, van Loosdrecht MCM (1997) Towards a more sustainable municipal wastewater treatment system. Water Sci Technol 35:171C180 Kampschreur MJ, Poldermans R, Kleerebezem R, van der Star WRL, Haarhuis R, Abma WR, Jetten MSM, van Loosdrecht MCM (2009a) Emission of nitrous oxide and nitric oxide from a fullscale single-stage nitritation-anammox reactor. Water Sci Technol 60: Kampschreur MJ, Temmink H, Kleerebezem R, Jetten MSM, van Loosdrecht MCM (2009b) Nitrous oxide emission during wastewater treatment. Water Res 43: Kartal B, Kuypers MMM, Lavik G, Schalk J, den Camp H, Jetten MSM, Strous M (2007) Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environmental Microbiology 9(3):635C642 Kartal B, Tan NCG, Van de Biezen E, Kampschreur MJ, Van Loosdrecht MCM, Jetten MSM (2010) Effect of nitric oxide on anammox bacteria. Appl Environ Microbiol 76: Kuai LP, Verstraete W (1998) Ammonium removal by the oxygenlimited autotrophic nitrification-denitrification system. Appl Environ Microbiol 64: Lackner S, Terada A, Smets BF (2008) Heterotrophic activity compromises autotrophic nitrogen removal in membrane-aerated biofilms: Results of a modeling study. Water Res 42: Loy A, Horn M, Wagner M (2003) probeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 31:514C516 Mancinelli RL, McKay CP (1983) Effects of nitric-oxide and nitrogendioxide on bacterial-growth. Appl Environ Microbiol 46:198C202 Metcalf, Eddy (2003) Wastewater engineering: treatment and reuse. McGraw-Hill, New York Pynaert K, Smets BF, Wyffels S, Beheydt D, Siciliano SD, Verstraete W (2003) Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor. Appl Environ Microbiol 69:Appl Microbiol Biotechnol Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63: Siegrist H, Salzgeber D, Eugster J, Joss A (2008) Anammox brings WWTP closer to energy autarky due to increased biogas production and reduced aeration energy for N-removal. Water Sci Technol 57:383C388 Starkenburg SR, Arp DJ, Bottomley PJ (2008) Expression of a putative nitrite reductase and the reversible inhibition of nitrite-dependent respiration by nitric oxide in Nitrobacter winogradskyi Nb-255. Environ Microbiol 10: Tchobanoglous G, Burton FL, Stensel HD (2003) Wastewater engineering, treatment and reuse―Metcalf & Eddy. McGraw-Hill, New York Third KA, Sliekers AO, Kuenen JG, Jetten MSM (2001) The CANON system (completely autotrophic nitrogen-removal over nitrite) under ammonium limitation: interaction and competition between three groups of bacteria. Syst Appl Microbiol 24:588C596 Tsushima I, Kindaichi T, Okabe S (2007) Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR. Water Res 41:785C794 Vazquez-Padin JR, Fernandez I, Morales N, Campos JL, MosqueraCorral A, Mendez R (2011) Autotrophic nitrogen removal at low temperature. Water Sci Technol 63: Verstraete W, Vlaeminck SE (2011) ZeroWasteWater: short-cycling of wastewater resources for sustainable cities of the future. Int J Sustain Dev World Ecol 18:253C264 Vlaeminck SE, De Clippeleir H, Verstraete W (2012) Microbial resource management of one-stage partial nitritation/anammox. Microb Biotechnol 5:433C488 Weissenbacher N, Takacs I, Murthy S, Fuerhacker M, Wett B (2010) Gaseous nitrogen and carbon emissions from a full-scale deammonification plant. Water Environ Res 82:169C175 Wett B (2006) Solved upscaling problems for implementing deammonification of rejection water. Water Sci Technol 53:121C128 Wett B, Buchauer K, Fimml C (2007) Energy self-sufficiency as a feasible concept for wastewater treatment systems. IWA Leading Edge Technology conference, Singapore Wett B, Nyhuis G, Hell M, Takacs I, Murthy S (2010) Development of enhanced deammonification selector. WEFTEC10, New Orleans Winkler MKH, Kleerebezem R, Kuenen JG, Yang J, van Loosdrecht MCM (2011) Segregation of biomass in cyclic anaerobic/aerobic granular sludge allows the enrichment of anaerobic ammonium oxidizing bacteria at low temperatures. Environ Sci Technol 45: Zart D, Schmidt I, Bock E (2000) Significance of gaseous NO for ammonia oxidation by Nitrosomonas eutropha. Anton Leeuw Int J Gen Mol Microbiol 77:49C55
更多搜索：
All rights reserved Powered by
文档资料库内容来自网络，如有侵犯请联系客服。