Effect of Low-Density Static Magnetic Field on the Oxidation of Ammonium by Nitrosomonas europaea and by Activated Sludge in Municipal Wastewater

Rapid population growth increases problems associated with water pollution, especially in municipal areas (1). The infl uent to municipal wastewater treatment plants contains high levels of both organic and inorganic nitrogen and mineralisation of the former results in the release of ammonium (2). Ammonium is a common pollutant that is normally eliminated from wastewater by two processes, nitrifi cation and denitrifi cation (3). Nitrifi cation is the biological oxidation of ammonia to nitrite, and subsequently to nitrate, by two groups of nitrifying bacteria. The fi rst group, ammonia-oxidising bacteria (AOB), obtain energy for growth by oxidising ammonium to nitrite and are usually represented in wastewater treatment plants by two genera, Nitrosomonas and Nitrosospira (2). The second group, nitrite-oxidising bacteria (NOB) ISSN 1330-9862 original scientifi c paper


Introduction
Rapid population growth increases problems associated with water pollution, especially in municipal areas (1).The infl uent to municipal wastewater treatment plants contains high levels of both organic and inorganic nitrogen and mineralisation of the former results in the release of ammonium (2).Ammonium is a common pollutant that is normally eliminated from wastewater by two processes, nitrifi cation and denitrifi cation (3).Nitrification is the biological oxidation of ammonia to nitrite, and subsequently to nitrate, by two groups of nitrifying bacteria.The fi rst group, ammonia-oxidising bacteria (AOB), obtain energy for growth by oxidising ammonium to nitrite and are usually represented in wastewater treatment plants by two genera, Nitrosomonas and Nitrosospira (2) (4).Both groups typically grow autotrophically, obtaining carbon by fi xation of CO 2 .Nitrosomonas europaea is a key AOB in the treatment of industrial and domestic wastewater; it plays an important role in the global nitrogen cycle (5)(6)(7)(8) and has been used as a 'model' AOB for physiological and other studies (9).
Ammonia oxidation limits the rate of ammonium removal from wastewater and is one of the most sensitive biological processes during wastewater treatment.It is highly sensitive to various environmental stresses including changes in pH, temperature and the presence of pollutants such as pharmaceuticals (10)(11)(12).AOB generally have low maximum specifi c growth rate, with doubling times in the range of 8 to 24 h, and their high sensitivity to environmental stresses makes their activity a critical step in biological nitrogen removal (13).Novel approaches for optimising ammonium removal are therefore in great demand and increasing att ention has been directed towards the potential for static magnetic fi elds (SMFs) to improve wastewater treatment (14).
Recent studies have demonstrated accelerated removal of organic substrates from artifi cial wastewater by static magnetic fi elds (SMF) (1,(14)(15)(16).The maximum substrate removal (up to 44 %) from glucose-based artifi cial wastewater was observed at an SMF of 17.8 mT (1).An SMF of 150-350 mT increased phenol degradation rate in bioreactors containing immobilised microbial beads (17) and formaldehyde degradation by activated sludge in artifi cial wastewater was increased by an SMF of 7 mT, decreasing formaldehyde concentration by up to 30 % (16).Under anaerobic conditions, SMF increased ammonium oxidation by an anammox consortium (18).However, despite the importance of aerobic nitrifi cation in activated sludge processes, the infl uence of SMF on aerobic ammonium-oxidising bacteria has not been investigated previously.
We have recently demonstrated that an SMF of 17 mT inhibited the growth of two wastewater bacteria, Escherichia coli and Pseudomonas putida, and increased their dehydrogenase activity and intracellular adenosine triphosphate (ATP) levels (19).Here we test the hypothesis that SMF also infl uences ammonia oxidation by the model AOB N. europaea.In addition, we examine aerobic ammonium removal from municipal wastewater by sludge bacteria in sequencing batch reactors (SBRs) during exposure to four diff erent SMFs (0, 10, 30 and 50 mT).Finally, we quantify the infl uence of SMF on the abundance of cultured N. europaea and sludge AOB in SBR.

Magnetic fi eld exposure
An SMF was generated by a cylindrical coil as described previously (19).Activated sludge in SBR was exposed to the SMF in the centre of the coil, where the magnetic density (B m ) was maintained at 0, 10, 30 or 50 (±0.5)mT.N. europaea cultures were exposed to the SMF in Petri dishes (90-mm diameter) on a nonconductive stand at B m of (17±0.5)mT.The SMF was continuously monitored by FH51 Gauss meter (Magnet-Physics, Dr. Steingroever GmbH, Köln, Germany).Exposed samples and unexposed controls were incubated in the dark at (21±0.5) °C, with temperature maintained by airfl ow (coil) or using a thermostatically controlled incubation chamber (coil and controls).All experiments with wastewater activated sludge and pure cultures were independently repeated three times.In addition, the experiments with pure cultures included triplicate cultures that were either exposed to the SMF (three biological replicates) or unexposed (three controls) within each independent experiment, giving nine exposed and nine unexposed experimental cultures for each experimental setup.

Bacterial strains, media and growth conditions
Frozen cultures of N. europaea Winogradsky (ATCC 19718, LGC Standards GmbH, Wesel, Germany) were inoculated into Erlenmeyer fl asks containing 150 mL of sterile mineral salts medium for ammonia oxidisers containing (NH 4 ) 2 SO 4 0.5 g/L (20).Cultures were incubated for four weeks at 28 °C and contamination was assessed periodically by inoculation of samples onto R2A agar plates (ATCC medium 1981, LGC Standards GmbH).

The infl uence of SMF on ammonium removal and bacterial abundance in batch cultures of N. europaea
A culture of N. europaea (15 mL) was inoculated into 150 mL of fresh AOB medium (containing NH 4 + 150 mg/L) and shaken at 200 rpm for 30 min at 28 °C, aft er which 20--mL samples were transferred to six Petri dishes.Three Petri dishes were exposed to the SMF for 7 days at B m =(17±0.5)mT and a temperature of (21±0.5)°C, and the three control Petri dishes were incubated under the same conditions, but not exposed to the SMF (only to the geomagnetic SMF).Ammonia oxidiser growth was monitored by determining the absorbance (A 650 nm ), ammonium and nitrite concentrations and viable cell concentration (most probable number, MPN).For MPN counts, serial dilutions were made in 96-well plates by transferring 40 μL of cell suspension to successive wells in microtiter plates containing 180 μL of sterile ammonia oxidiser medium (20).Growth was assessed by the presence of nitrite with modifi ed Griess-Ilosvay reagents aft er incubation at 28 °C for four weeks.MPN per mL of culture was estimated using the BAM-MPN Excel spreadsheet calculator (US FDA, Silver Spring, MD, USA).

Sampling of activated sludge and wastewater
Activated sludge and municipal wastewater were obtained from a municipal wastewater treatment plant (Ptuj Municipal Service Corporation, Slovenia) on February 9, 2012.Activated sludge was removed from 150 L of municipal wastewater by sedimentation, and wastewater without the active biomass was stored in 1.5-litre bott les at -20 ˚C until use.Municipal wastewater at the time of sampling contained (in mg/L): NH 4 + -N 25.8, NO 3 --N 0.13, total nitrogen (TN) 26.8 and total organic carbon (TOC) 35.9.Nitrite was below the detection limit (0.452 mg/L).Fresh activated sludge was taken from the aeration basin of the Ptuj Municipal Service Corporation before each experiment and was added to previously stored wastewater aft er acclimatisation for four days.The fi nal mixture used for all experiments contained 300 mL of fresh activated sludge (mixed liquor suspended solids (MLSS) 5.7 g/L) and 1700 mL of stored municipal wastewater.

The infl uence of SMF on ammonium removal from wastewater in sequencing batch reactors
The infl uence of SMF on ammonium removal was investigated in two sequencing batch reactors (SBR) with working volumes of 2 L, one with magnetic fi eld accessories (experimental samples) and the other without magnetic modifi cation (control samples).The SBRs were inoculated with 300 mL of fresh activated sludge (MLSS, 5.7 g/L) and were exposed to SMF of 10, 30 or 50 (±0.5)mT or unexposed (control bioreactor) for four days at (21±0.5) °C.During this acclimatisation period, which allowed adaptation of microorganisms in activated sludge to the new conditions, pH was maintained at 7.8±0.2and dissolved oxygen (DO) at (3.0±0.3)mg/L.Acclimatised sludge was then mixed with 1700 mL of stored municipal wastewater, and the system continued to be operated at the same conditions: 0, 10, 30 or 50 mT, temperature of (21±0.5)°C, and sampled hourly for 25 h to analyse ammonium and nitrate concentrations and ammonia oxidiser MPN.The experiment was repeated independently three times.
The ammonium oxidation rate (q N ) was expressed in mg of NH  (24) for TN, following oxidation of nitrogen oxides, using the TOC analyser equipped with a TN measuring unit TOC-VCPN (Shimadzu, Kyoto, Japan).MLSS were measured according to standard methods (APHA method 2540 D) (25).The pH and DO concentration were measured using a digital, portable pH and DO meter (HQ 40d multimeter, Hach-Lange, Loveland, CO, USA), respectively.

Statistical analysis
Data analysis and graphical representation were performed using Excel (Microsoft , Redmond, WA, USA).Comparisons between treatments were performed using a two-sample Student's t-test assuming equal variance (differences were considered signifi cant at p<0.05).Values are reported as mean±standard error of nine independent replicates for experiments with N. europaea and for nine independent replicates for experiments in the SBR bioreactors.

Results and Discussion
The infl uence of SMF on ammonia oxidation and growth of N. europaea Removal of ammonium nitrogen in wastewater treatment plants is limited by the rate of ammonia oxidation by AOB.This work shows for the fi rst time that SMF, at 17 mT, signifi cantly stimulated the removal of ammonia by the model chemolitotrophic wastewater AOB N. europaea.Cultures of N. europaea were exposed to an SMF of 17 mT, which had previously been found to infl uence growth and physiology of E. coli and P. putida (19).Ammonium and nitrite concentrations initially decreased and increased, respectively, in a similar manner in both exposed and unexposed samples and no infl uence of SMF was observed (Fig. 1).This initial increase in nitrifi cation was presumably due to the transfer of N. europaea cultures into the fresh medium where ammonium and oxygen were abundant.Ammonium and nitrite did not change significantly in exposed or unexposed cultures during the fi rst fi ve days.Aft er this transient lag phase, ammonium oxidation resumed with a marked diff erence between the exposed and unexposed cultures, such that SMF exposure for 7 days resulted in (30±4) % (p=0.037)increase in ammonium removal and a corresponding (26±5) % (p=0.042)increase in nitrite concentration, in comparison with unexposed controls (Fig. 1).Dehydrogenase activity in Escherichia coli and Pseudomonas putida was also stimulated by SMF at 17 mT, but stimulation increased within 2 h of exposure and a prolonged lag phase was not detected (19).A rapid depletion of oxygen might have caused the lag phase observed in the experiments with N. europaea, as nitrifi cation is sensitive to oxygen fl uctuations (26).SMF of 17 mT also stimulated growth of N. europaea, which was, like stimulation of ammonium oxidation, detected only aft er fi ve days of incubation.The ratio of N. europaea MPN counts in exposed and unexposed cultures was not signifi cantly diff erent on days 1 (p=0.071)or 3 (p=0.060),but increased to 1.4 (p=5.4•10-10 ) on day 5 and to ) on day 7 (Fig. 2), when stimulation of ammonium oxidation was also observed.It is possible that only actively growing cells are sensitive to magnetic fi eld, consistently with our previous work (19) showing that the eff ect of magnetic fi eld on heterotrophic bacteria is most prominent in conditions supporting the highest growth rates.Although SMF stimulated the growth of N. europaea, in contrast to the inhibition of growth of E. coli and P. putida (19), in both experiments cultures were exposed to the same SMF density.The diff erent responses, inhibition vs. stimulation, may be due to the diff erent metabolic and energy-generating pathways of heterotrophs and a chemolithotroph and the much slower growth of N. europaea.

The infl uence of SMF on ammonium removal from municipal wastewater and on abundance of AOB in activated sludge
Stimulation of N. europaea growth and nitrifi cation activity in cultures by SMF suggested the potential for stimulation of nitrifi cation in wastewater treatment processes, which was tested using municipal wastewater and wastewater sludge bacteria.The infl uence of four diff er-SMF densities (B=0, 10, 30 or 50 mT) on the removal of NH 4 + -N from municipal wastewater was studied in SBRs at constant temperature of (21±0.5)°C.The results presented in Fig. 3 and Table 1 indicate an initial lag phase with very low ammonium oxidation rates for approx. 2 h in both exposed and unexposed cultures.Ammonia oxidation then increased in all treatments but at signifi cantly higher rates in SBRs exposed to 50 or 30 mT than in control bioreactors (Table 1).The strongest increase of (43±2) % at 2-8 h and (77±5) % at 8-12 h was detected in SBRs exposed to 50 mT, compared to the unexposed SBRs.In contrast, exposure to 10 mT reduced the ammonium removal rate to (0.12±0.03) mg of NH 4 + -N per g of MLSS per hour, which was (40±3) % lower than the control rate aft er exposure for 12-25 h (Table 1).SMF therefore had positive or negative eff ects on ammonium removal rates from wastewater in SBRs, depending on the density of the applied magnetic fi eld.A positive and negative response to SMF of diff erent strengths has also been observed in other processes.For example, anammox process was stimulated at the SMF of 30 mT but inhibition of this process occurred at 110 mT (18).The infl uence of SMF therefore depends on the strength of the applied SMF but the specifi c response to a particular SMF density depends also on the process that is studied.This work also provides the fi rst evidence that SMF increases the growth of sludge AOB.The abundance of AOB in wastewater SBRs exposed to diff erent SMF densities (0, 10, 30 and 50 mT) was determined by MPN aft er exposure for 25 h following the four-day acclimatisation phase of the activated sludge.AOB abundance increased signifi cantly at 50 mT (1.4-fold; p=2.8•10 -13 ) and at 30 mT (1.2-fold; p=1.2•10 -8 ), but not at 10 mT (p=0.1), when compared to the unexposed samples (Fig. 4).An increase in AOB abundance may in part be responsible for higher removal rates measured in SBRs exposed to 30 or 50 mT but Several mechanisms infl uencing the growth and/or enzyme activities may be responsible for the observed effects.One of the most intensely studied enzymes in relation to the eff ects of magnetic fi eld is B12-dependent ethanol ammonia lyase for which a decrease in v max /K m at 100-mT magnetic fi eld density was reported (27).It is known that enzymatic reactions that are responsive to magnetic fi elds must generate paramagnetic particles or spin correlated radicals (28)(29)(30).Interestingly, the hydroxylamine oxidase-associated tetraheme cyt cm552 from N. europaea, which catalyses the oxidation of hydroxylamine to nitrite, was reported to carry paramagnetic heme species (31).However, many enzymes with radical intermediates will not produce magnetic fi eld-dependent reaction kinetics and further studies are needed to test this hypothesis.Another possibility is changes in transcriptional activity of genes involved in ammonia oxidation, as SMF has shown to infl uence transcriptional activity of various genes (32,33).Although the mechanisms behind the observed phenomena are not understood and require further study, the work clearly shows that SMF stimulates ammonium removal by mixed activated sludge bacteria and by the pure culture of an important wastewater nitrifi er.

Conclusions
This work shows for the fi rst time that SMF positively infl uenced ammonia oxidation and the growth of an important and very sensitive wastewater AOB, Nitrosomonas europaea in the laboratory pure culture.Results showed that SMF of 17 mT increased ammonium removal for (30±4) % and nitrite removal for (26±5) % and also stimulated growth of N. europaea.Additionally, in the experiments with activated sludge and municipal wastewater, we showed that SMF of 50 mT increased ammonium removal rates by activated sludge bacteria in SBRs by up to 77 % and increased the abundance of sludge AOB.In con-trast, SMF of 10 mT decreased the activity by up to 40 % but did not infl uence the growth of bacteria in SBRs.These fi ndings may have important implications in the fi eld of biological wastewater treatment and suggest potential strategies for the removal of mineral nitrogen and growth manipulation of key nitrifi ers in municipal wastewater treatment plants.

Fig. 1 .
Fig. 1.The eff ect of static magnetic fi eld (SMF) of 17 mT on the oxidation of ammonia to nitrite by Nitrosomonas europaea.Cultures were exposed to SMF for 7 days.The error bars represent standard error of the mean (N=9). and : concentrations of NH 4 + -N (mg/L) in exposed and unexposed cultures, respectively;  and : concentrations of NO 2 -N (mg/L) in exposed and unexposed cultures, respectively

Fig. 3 .Fig. 2 .
Fig. 3.The eff ect of static magnetic fi eld (0, 10, 30 or 50 mT) on ammonium removal from wastewater by activated sludge during exposure for 25 h at 21 °C, plott ed as the ratio of ammonium concentration aft er the period (t) of exposure (c) to the initial concentration (c o ).The error bars represent standard error of the mean (N=3).Sequencing batch reactors were exposed to 0 (), 10 (), 30 () and 50 mT (•)

Fig. 4 .
Fig. 4. The eff ect of static magnetic fi eld (SMF) of 10, 30 and 50 mT on the ratio of viable cell concentration (determined by most probable number, MPN) of ammonia-oxidising bacteria (AOB) in activated sludge.The error bars represent standard error of the mean (N=3).Dashed line (no infl uence of SMF) indicates a ratio of one.Values that are above the dashed line indicate a positive infl uence of the SMF.*indicates a ratio signifi cantly different from 1 (p<0.05)

Effect of Low-Density Static Magnetic Field on the Oxidation of Ammonium by Nitrosomonas europaea and by Activated Sludge in Municipal Wastewater
. The second group, nitrite-oxidising bacteria (NOB) are represented by Nitrobacter and Nitrospira and oxidise nitrite to nitrate