Study of SBR Reactor Performance to Enrichment of Ammonium Oxidizing Organism in Moderate Weather

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1^th International Conference on Water, Environment and Sustainable Development, 27-29 September, 2016

University of Mohaghegh Ardabili, Ardabil, Iran

Study of SBR Reactor Performance to Enrichment of Ammonium Oxidizing Organism in Moderate Weather

Dr. Adnan Abbas Al-Samawi

¹

and Dr. Mohammed Siwan Shamkhi

²

1- Professor Emeritus of Environmental Engineering, University of Technology, Iraq 2- Assistant Professor of Water Resources Engineering, Wasit University, Iraq

[email protected] Abstract

This study includes two parts; in the first part, enrichment of high active ammonium oxidizing organisms (AOO) in sequencing batch reactor (SBR) in moderate weather and without temperature control , while in the second part; consideration of the influence of major operational parameters including; temperature, pH, dissolved oxygen concentration (DO) , and free ammonia (FA) and free nitrous acid (FNA) formation to evaluate the process performance in moderate weather without temperature control are investigated.

Results revealed that the maximum removal efficiencies of ammonium from synthetic reject water in the SBR inoculated with mixed liquor activated reactor was within 71.09%. The NAR had with a maximum value of 95.74% which refers to strengthen of growth of AOO. The specific ammonium oxidation rate was with 0.085 mg NH4+

-N/mgVSS.h which refer to high active enriched AOO and feasibility of enrichment of high activity nitrifiers in moderate weather.

Keywords: nitrifier, enrichment, SBR, moderate, weather.

1. INTRODUCTION

Partial nitrification is the oxidation of wastewater ammonium to nitrite but not to nitrate. To achieve partial nitrification, the subsequent oxidation of nitrite to nitrate must be prevented [1], [2]. For successful implementation of these technology, the critical point is how to maintain partial nitrification of ammonium to nitrite [3]. According to Monod equation a high substrate concentration is capable of promoting the microbial activity. So the AOO activity can be elevated by means of increasing substrate concentration with reactor condition help to avoiding FA inhibition, therefore high activity nitrifiers are considered as the key to high- rate partial nitrification process [4]. This paper aims to design of a selector reactor where in AOO predominate over the NOO is because the growth rates of the AOO are greater than the NOO at temperatures above 15¡C, and especially between 30-40¡C as shown in Figure (1) [5]. pH control will be a tool to get more stable partial nitrification and higher nitrite accumulation because pH control allowed operation at an optimum and inhibiting free ammonia FA concentration, for AOO and NOO, respectively [6], [7], [8], [9], [10].

Figure 1. Minimum HRT for growth of AOO and NOO as a function of temperature. (Van Kempen)

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2. M

ETHODS

2.1. EXPERIMENTAL SETUP

The nitrifying organism acclimation process has been developed at natural laburatory temperature The reactor have diameter of 15cm and total height of25cm. It was constructing of Perspex cylindrical with 3 liters work volume of and which able to adjusted to operate at minimum volume of 1.0 liter (which is the residual volume at the end of each SBR cycle). as shown in Figure (2). Fill – react – settle – and draw mode following a predefining cycle and operated continuously. During react phases, a stirrer device kept the reactor contents under homogenous condition at all times. Aerobic condition was achieved by compressed air. Filling, purging and extraction events were achieved by three electrical valves. The biomass was not removed from reactor. During extraction periods, treated reject water was discharge from the reactor until predefined minimum reactor water level was reached. The SBR was operated by electrical board control supplied with timers which apple to repeat over previously defined time cycle of operation by controlling the switching on/off of filling , the purge and draw electrical valves as well as mixing device and the air pumps.

The inoculums used was taken from Rustamiya WWTP (south of Baghdad) aerobic reactor (mixed liquor) which mostly contain heterotrophic bacteria, that remove organic carbon aerobically, and a small part of autotrophic bacteria that remove ammonium in presence of inorganic carbon. The average percentage of nitrifying bacteria in secondary activated sludge about 0.5-1% of mixed culture [11]. In order to develop the nitrifying organism, initially, influent concentration of 50 mg NH4+- N/l was supplied with HRT of 1.0 day.

Ammonium concentration was increased stepwise up to 500mg/L through the operation time after having an entire removal of the supplied ammonium.

Figure 2. Schematic diagram of enrichment of nitrification organism SBR experimental setup.

2.2. ANALYTICAL METHODS

Manually three time per day measurement of pH, dissolved oxygen and temperature. Manually acid and base (H2SO4 and 1M NaHCO3) doses were added to keep the reactor with required pH. Two times per week, ammonium nitrogen NH4+-N, nitrite nitrogen NO2--N, and nitrate nitrogenNO3--N was analyzed by spectrophotometric method.

3. R

ESULTS

The performance of the enrichment phase of ammonium oxidizing organism (AOO) was evaluated mainly in terms of the ammonium oxidization to nitrite and nitrate as well as the volatile suspended solid (VSS).

3.1 PHYSICAL PARAMETERS

The variation of temperature, pH, and conductivity were presented in Figures 3 to 5. As shown in Figure 3, it can seen that the temperature in SBR reactor for enrichment of AOO was kept around 29 �0.07 ᵒC which was

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natural temperature of laboratory without using additional heating therefore the fluctuation was appear during whole enrichment period. The temperature was with optimal range to improve the AOO than nitrite oxidizer organisms (NOO) due to the fact that the activation energy of the ammonia oxidation step is higher than that of the nitrite oxidation step. Thus, nitrification proceeds better in warmer seasons or climates. as proven by [12], [13], 14], [15]. Also, an increase in temperature causes an increase in FA concentration. The rising temperatures affect AOO and NOO via the formation of FA that is more inhibitory to NOO as reported by [16].

The pH profile in Figure 4, demonstrates that the influent pH was stable with an average value of 8

�0.04; however, the pH inside the SBR reactor dropped down to 7 �0.07. The decrease in the pH as a result of the alkalinity consumption and the H⁺ion release implied that the AOO were very active and a high amount of nitrite was produced because the pH value in the system affected the nitrifying activity due to its effect on the NH₃/NH₄⁺ and the HNO₂/NO₂^- equilibrium which concur with the previously reported value by [14], [17]. The pH was corrected periodically to be around neutral by adding NaHCO3 because pH is the key parameter in driving the nitrification process as was proven by [6], [7], [5], [15], and to avoid free nitrous acid formation.

Figure 3. variation of temperature during AOO enrichment period.

Figure 4. variations in the pH during the AOO enrichment period.

The average concentration of dissolved oxygen was maintained around 1.62 � 0.04 mg/l. The DO concentration concurred with the value recommended for the nitrification by [18].

Figure 5 shows that the electrical conductivity of the influent reject water increases as the ammonium concentration increases, because the electrical current is transported by the ions in solution;

therefore, the increase in the NH4+ and HCO3- ion concentrations which are the main compositions of the synthetic reject water increases the conductivity as the concentration of the ammonium ion increases. The average electrical conductivity values of the influent reject water was 1.13� 0.12 mS/cm, while the average values of the electrical conductivity of the effluent reject water was 1.35�0.12 mS/cm. The increase in the effluent electrical conductivity indicates that the sum of the absolute values of the charges for the H⁺, NO2-

and NO3- ions released during nitrification process by the enriched AOO plus the values of the charges of the residual NH4+ and HCO3- was greater than the sum of the absolute value of the charge of the influent.

3.2 CHEMICAL ANALYSIS

The chemical analyses of the influent and effluent in the SBR reactor during the enrichment of the stage of the ammonium oxidizing organisms showed that the effluent nitrite and nitrate concentrations recorded an average 48�12 mg/l and 18� 3.0 mg/l, respectively. This result refers to the growth of both the ammonium oxidizing organisms (AOO) and nitrite oxidizing organisms (NOO) in SBR reactor, as shown in Figure 6;

however, the nitrite concentration was higher than the nitrate concentration, which means that the biomass concentration of the AOO was greater than the biomass concentration of the NOO in the SBR reactor for the enrichment of the AOO. The growth of the nitrite oxidizing organisms in the enrichment SBR reactor under the reactor conditions which enhanced the growth of the nitrite oxidizing organisms, such as temperature, was in the range which enabled the growth of these two kinds of organisms (see Figure 1). The concentration of the dissolved oxygen in the SBR reactor was at an average value of 1.62 �0.04 mg/l, which enabled complete nitrification to be achieved, was similar to the previously published values of 1.5 mg/l [19], 1.7

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mg/l [20] and 1.4mg/l [21].Therefore, the reduction of the nitrate concentration in the SBR reactor of the ammonium oxidizing organisms was due to the inhibition of the nitrite oxidizing organisms by the Free Ammonia (FA) concentration formed in the reactor, whereas the nitrite oxidizing organisms inhibited with 0.1 – 1.0 mg/l of FA, while the ammonium oxidizing organism inhibited with 10 – 150 mg/l FA, as reported by [22].

Figure 5. variations in the EC during the AOO enrichment period.

Figure 6. Nitrite and nitrate concentrations during the period of the stage of the enrichment

by the ammonium oxidizing organisms.

Figure 7 shows the concentration of the FA and free nitrous Acid (FNA) in the SBR reactor.

Inhibition of the FNA did not occur due to the significant accumulation of nitrite in the reactor because the concentration of the FNA was below 0.2 mg/l during the operative period, which concurs with [23]. The concentration of the FA was observed to be 1.5 � 0.48 mg/l while the maximum concentration was 9.64 mg/l. As shown in Figure 8 and Figure 9 the nitrite was starting to accumulate and the nitrate concentration decreased when the FA appeared which is in agreement with the results previously reported by [8], [10].

Also, it was very clear that the NOO was inhibited whereas the nitrate concentration reaches to near zero when the FA increase up to nearly 10 mg/l; then the nitrate again appeared when the FA decreased to minimum values. This relationship between the nitrate formation and FA concentration indicates that the FA inhibits the NOO but does not kill it. However, this finding is in agreement with the results previously reported by [1].

3.2.1

AMMONIUMREMOVALEFFECIENCYANDNITRITEACCUMULATIONRATIO

The variations in the concentrations of the influent and effluent ammonium nitrogen are presented in Figure 8 , where the concentrations of the influent and effluent ammonium showed an average of 116 � 15 mg/l and 44� 4 mg/l, respectively. The maximum removal efficiency of the ammonium in the SBR reactor for the enrichment of the AOO was 71% and the average value from start up to the end was 50 3% . To investigate the capability of strengthening the growth of the AOO and suppressing the growth of the NOO, the nitrite accumulation ratio (NAR) was calculated with following equation given by [24]:

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The maximum value of NAR was 96% with the average value from the commencement of the SBR reactor operation until the end of the operation and the average value of the NAR was 50 7%. As shown in Figure 9, the NAR value at the beginning of the SBR operation was very low with a minimum value reaching zero. The reason for this increase in the NAR values was due to the reactor conditions of temperature and pH and the formation of the FA, as demonstrated earlier and shown in Figure 10. Previous studies conducted by [1], [8], [13], [19], [23], [25] reported that the DO limitation is the main factor used to control the nitrite accumulation. The NAR result did not agree at this point whereas the NAR reached to 95.74% with the DO showing an average concentration of 2.64� 0.23 mg/l, but concurred with the earlier study of [24].

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Figure 7. Free ammonia FA and free nitrous acid (FNA) concentration during the enrichment period

by the ammonium oxidizing organisms.

Figure 8. Variations in the ammonium concentration during the enrichment stage of the ammonium oxidizing organisms.

3.3 HYDRAULIC RETAINTION TIME (HRT)

The effect of the Hydraulic Retention Time (HRT) on ammonium removal in the SBR reactor for the enrichment of the AOO was evaluated by the operation of the reactor at three different HRTs as shown in Figure 10. It was observed that the HRT increased from 0.955 day to 1.82 days. The removal efficiency of the ammonium increased from 7 % to 71%. The difference between the minimum and maximum values of the percentage of the ammonium removal being very low with HRT equal to 1.82 days. This minimum difference was attributed to the optimum HRT (1.82 d) corresponding to the average temperature which promoted the stability of the nitrification process which decreased the differences between the minimum and maximum ammonium removal efficiencies. Therefore, the operation of the SBR reactor continued with this value of the HRT to the end of the AOO enrichment phase as the optimum HRT. After one day of increasing the HRT from 1.068 days to 1.2 days (at time 13 days), the nitrite accumulation commenced while the nitrate concentration was decreased, as shown in Figure 6 which ensured the washout of the nitrite oxidizing organisms (NOO). The nitrite accumulation continued to increase when the HRT increased to 1.82 days, which indicated the importance of the ammonium loading rate parameter, given by equation 2 to evaluate the nitrite accumulation rate as shown in Figure11, with a correlation of 0.92.

3.4 CHARECTERISTICS OF AMMONIUM OXIDIZING ORGANISMS (AOO) 3.4.1 SPECIFIC AMMONIUM OXIDATION RATE

The specific ammonium oxidation rate describes the affinity of the AOO. In other words, the activity of the nitrifiers was represented by the specific ammonium oxidation rate, which is the substrate utilization rate per VSS concentration. Figure 12, demonstrates the gradual increase of the specific ammonium oxidation rate for AOO enrichment in the SBR reactor, which indicates that the activity of the nitrifiers is high.

Figure 9. Variations in the nitrite accumulation rate Figure 10. The effect of HRT on the % of removal of Ammonium loading rate = (2)

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University of Mohaghegh Ardabili, Ardabil, Iran .

Figure 11. The correlation between the ammonium loading rate and nitrite accumulation rate

Figure 12. The variation in the specific ammonium oxidation rate.

As shown in Figure 13, the specific ammonium oxidation rate was directly related to the concentration of the influent ammonium. The maximum specific ammonium oxidation rate observed was 0.085 mgNH₄⁺--N/mgVSS.h, which refers to the highly active AOO that was enriched compared with that reported by [15] for the conventional activated sludge process in which the range was between 0.0019 and 0.015 mgNH₄⁺--N/mgVSS.h. The values obtained compared well with the trend of the specific growth rate obtained by the researchers from the bioreactors. The results were in good agreement with the results previously reported by [4], [6], [21], [26], [27]. According Chen at al., (2010) [4], who defined the AOO as high activity nitrifiers, when the maximum specific ammonium oxidation rate was above 1.8 mgNH₄⁺-- N/mgVSS.d (0.075 mgNH₄⁺--N/mgVSS.h) the enriched AOO can be termed high activity nitrifiers.

3.4.2 YIELD OF THE AMMONIUM OXIDIZING ORGANISMS

The AOO yield, described as the mass of the new AOO cells produced per unit of ammonium removed was 0.29, as shown in Figure 14. The yield value was low compared with the value reported for the AOO by [17].

The decay rate of the AOO which represented the rate of decrease of the AOO due to the death of some of the AOO cells to the AOO concentration in the reactor was 0.097 d^-1, as shown in Figure The decay rate value was high compared with the value reported by [17]. To evaluate the value of the AOO yield obtained, it was compared with the value of the AOO yield calculated based on bioenergetics.

The stoichiometry of the biological reactions can be expressed as:

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Figure 13. The relationship between the specific ammonium oxidation rate and ammonium

concentration.

Figure 14. Determination of bio-kinetic constants (cell yield and decay rate)

Where R, R_a, R_cs and R_d are the overall balance reaction, half reaction of the electron acceptor, half reaction of the cell tissue synthesis and the half reaction of the electron donor, respectively. Also, fe and fs are

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the fractions of the electron donor used for the energy and fraction of the electron donor used for cell synthesis, respectively. The half reactions describe the transfer of 1 mole of the electron in the oxidation and synthesis reactions .The half reactions of oxygen as the electron acceptor, cell synthesis and ammonium oxidation as the electron donor are given in equations 4, 5 and 6 below:

(4) (5) (6) the balance stoichiometric reaction of the oxidation of ammonium can be given as follows:

(7) So,

(8) From equation (8),

The ammonium utilized for cell production =

The cell yield determined by:

then (9) According to Rittmann and McCarty (2001) [28], the normal value of f_e and f_s are 0.86 and 0.14,

respectively. Following equation (10), the amount of the biomass produced during cell synthesis relative to the amount of the substrate degraded which is called ‘true yield’ can be calculated as 0.326 gmVSS/gmNH4+- -N, which is more than the net yield that was determined by the kinetics experiments. The reason for the low yield and high activity of the enriched AOO cannot be explained directly from the previous results. The results can give us an indication that the fraction of electron donor used for cell synthesis f_s is less than the normal value, which was given earlier. Also, the fraction of the electron donor used for energy f_eis more than the normal value given prior. These finding can be explained in the same way that Chen et al., (2010) [4] did, who stated that the enriched high activity AOO consumed higher energy for cell maintenance than for cell synthesis. Also, the high endogenous decay rate was referred to the loss in cell mass due to the oxidation of the internal storage products for the energy required for cell maintenance.

4. CONCLUSIONS

The AOO was enriched in the SBR reactor successfully at a moderate laboratory temperature of 29.0±0.07ᵒC. The AOO growth was strengthened because the NAR was at its maximum value of 95.74%.

The correlation of 0.92 between the nitrite accumulation rate and ammonium loading rate is an important parameter to evaluate the nitrite accumulation. The specific ammonium oxidation rate at 0.085 mgNH₄⁺-- N/mgVSS.h which refers to the highly active AOO was enriched and the feasibility of the enrichment of the high activity nitrifiers in moderate weather, such as the weather of southern of Iraq. The yield of the enriched AOO was 0.29 d^-1which was low compared with the yield calculated based on the bioenergetics and decay rate of the AOO was 0.097 d^-1, which is high. Therefore, the enriched AOO was classified as a high activity nitrifier. The high activity nitrifiers consume high energy for cell maintenance than for cell synthesis. The relation between nitrate formation and the FA concentration indicates that the FA inhibits the NOO but does not kill them because the nitrate was seen to disappear when the FA increased up to 10mg/l and then appeared again when the FA dropped to 0.05mg/l.

5. ACKNOWLEDGMENT

My sincere appreciation is forwarded to all staff of Industurial Waste water Treatment Plant in Kut textile factory (Iraq) for their help in laboratory tests.

The cells produced =

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6. REFERENCES

1.Peng, Y. and Zhu, G., (2006), “Biological nitrogen removal with nitrification and denitrification via nitrite pathway”, Appl. Microbiol. Biotechnol., Vol. 73, pp. 15–26.

2.Schmidt, I., Sliekers, O., Schmid, M., Bock, E., Fuerst, J., Kuenen, J. G., Jetten, M. S., Strous,M, (2006),

“New concepts of microbial treatment process for the nitrogen removal in wastewater”, FEMS Microbiology Reviews, Vol. 27, pp. 481-492.

3.Sliekers, A. O, Derwort, N., Gomez, J. L.C., Strous, M., Kuenen, J. G., Jetten, M.S.M., (2002),

“Completely autotrophic nitrogen removal over nitrite in one single reactor”, Water Research, Vol. 36 , pp.

2475–2482.

4.Chen, J., Zheng, P., Yu, Y., Mahmood, Q., Tang, C., (2010), “Enrichment of high activity nitrifers to enhance partial nitrification process”, Bioresource Technology, Vol. 101, pp. 7293–7298.

5.Van Kempen, R., Mulder, J. W., Uijterlinde, C. A., Loosdrecht, M.C.M., (2001), “ Overview: full scale experience of the SHARON process for treatment of rejection water of digested sludge dewatering”, Water Science and Technology, Vol. 44, No. 1, pp. 145–152.

6.Ciudad, G., Gonza, R., Bornhardt, C., Antileo, C., (2007), “Modes of operation and pH control as enhancement factors for partial nitrification with oxygen transport limitation”, Water Research, Vol. 41, No. 20, pp. 421-4629.

7.Ishak, A. A. and Hussain, M. A., (2003), “Review of Critical Ranges in pH Control for Bioprocesses”, 2nd Technical Postgraduate Symposium, TechPos’03.

8.Yang, J., Zhang, L., Daisuke, H., Takahiro, S., Ma, Y., Li, Z., Furukawa, K, (2010), “ High rate partial nitrification treatment of reject wastewater”, Journal of Bioscience and Bioengineering, Vol. 110, No. 4, pp. 436–440.

9.Park S. and Bae, W., (2009), “Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia and free nitrous acid”, Process Biochemistry, Vol. 44, pp. 631–

640.

10.Xue, Y., Yang, F., Liu, S., Fu, Z., (2009), “ The influence of controlling factors on the start-up and operation for partial nitrification in membrane bioreactor”, Bioresource Technology, Vol. 100, pp. 1055–

1060.

11.Metcalf & Eddy, Inc., ( 2003), “ Wastewater engineering: treatment, disposal and reuse”, McGraw-Hill.

12.Vazquez-Padin, J. R., Figueroa, M., Campos, J. L., Mosquera-Corral, A., Méndez, R., (2010), “Nitrifying granular systems: A suitable technology to obtain stable partial nitrification at room temperature”, Separation and Purification Technology, Vol. 74, pp. 178.

13.Jubany, I., Lafuente, J., Baeza, J. A., Carrera, J., (2009), “Total stable washout of nitrite oxidizing bacteria from nitrifying continuous activitaed sludge system using automatic control base on oxygen uptake rate measurements, Water reaserch , Vol. 43, pp. 2761-2772.

14.Yan, J. and Hu, Y. Y., (2009), “Partial nitrification to nitrite for treating ammonium-rich organic wastewater by immobilized biomass system”, Bioresource Technology, Vol. 100, pp. 2341–2347.

15.Hellinga, C., Schellen, A. A. J. C., Mulder, J. W., van Loosdrecht, M. C. M., Heijnen, J. J., (1998), “ The Sharon process: an innovative method for nitrogen removal from ammonium rich wastewater”, Water Science and Technology, Vol. 37, pp.135-142.

16.Kim, D. J., Lee, D., I., Cha, G. C., Keller, J., (2008), “Analysis of Free Ammonia Inhibition of Nitrite Oxidizing Bacteria Using a Dissolved Oxygen Respirometer”, Environ. Eng. Res., Vol. 13, No. 3, pp. 125- 130.

17.Dinҫer, A. and Kargi, F., (2000), “Kinetics of sequential nitrification and denitrification processes”, Enzyme and Microbial Technology, Vol. 27, pp. 37–42.

18.EPA, (1993), “ Manual nitrogen removal”, EPA/625/R-93/010.USA.

19.Garrido, J. M., van Benthum, W. A. J., van Loosdrecht, M. C. M., Heijnen, J. J., (1996), “Influence of Dissolved Oxygen Concentration on Nitrite Accumulation in a Biofilm Airlift Suspension Reactor”, Biotechnology and Bioengineering, Vol. 53, No. 2, pp. 167-178.

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20.Ruiz, G., Jeison, D., Chamy, R., (2003), “Nitrification with high nitrite accumulation for the treatment of wastewater with high ammonia concentration”, Water Research, Vol. 37, pp. 1371–1377.

21.Ciudad, G., Rubilar, O., Munoz, P, Ruiz, G., Chamy, R., Vergara, R., Jeison, D., (2005), “Partial nitrification of high ammonia concentration wastewater as a part of a shortcut biological nitrogen removal process”, Process Biochemistry, Vol. 40, No. 5, pp. 1715-1719.

22.Sinha, B. and Annachhatre, A. P., (2007), “Partial nitrification—operational parameters and microorganisms involved”, Rev. Environ. Sci. Biotechnol., Vol. 6, pp. 285–313.

23.Aslan, S., Miller, L., Dahab, M., (2009), “Ammonium oxidation via nitrite accumulation under limited oxygen concentration in sequencing batch reactors”, Bioresource Technology, Vol. 100, pp. 659–664.

24.Guo J. H., Peng, Y. Z., Wangb, S., Zheng, Y., Huang, H., Wang, Z. W., (2009), “Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure”, Bioresource Technology, Vol. 100, pp. 2796–2802.

25.Ahn,J. H.,Kwan, T. and Chandran, K., (2011), “Comparison of Partial and Full Nitrification Processes Applied for Treating High-Strength Nitrogen Wastewaters: Microbial Ecology through Nitrous Oxide Production”, Environ. Sci. Technol., Vol. 45, pp. 2734–2740.

26.Kim, J. H., Guo, X., Behera, S. K., Park, H. S., (2009), “A unified model of ammonium oxidation rate at various initial ammonium strength and active ammonium oxidizer concentrations”, Bioresource Technology , Vol. 100, pp. 2118–2123.

27.Guven, D., Kutlu, O., Insel, G., Sozen, S., (2009), “Model-based process analysis of partial nitrification efficiency under dynamic nitrogen loading”, Bioprocess Biosyst. Eng., Vol. 32, pp. 655–661.

28.Rittmann, B.E. and McCarty, P.L., (2001), “Environmental Biotechnology: Principles and Applications”, McGraw-Hill Companies.