EVALUATION OF AMMONIA REMOVAL IN WASTEWATER TREATMENT

USING CONTINUOUS AERATION AND INTERMITTENT AERATION

A Thesis

Presented to the faculty of the Department of Civil Engineering

California State University, Sacramento

Submitted in partial satisfaction of

the requirements for the degree of

MASTER OF SCIENCE

in

Civil Engineering

by
Zhengjian Yang

SUMMER
2019
 © 2019

Zhengjian Yang

ALL RIGHTS RESERVED

ii
 EVALUATION OF AMMONIA REMOVAL IN WASTEWATER TREATMENT

USING CONTINUOUS AERATION AND INTERMITTENT AERATION

A Thesis

by

Zhengjian Yang

Approved by:

__________________________________, Committee Chair

Dr. Amir Motlagh

__________________________________, Second Reader

Dr. Yujie Jin

____________________________
Date

iii
Student: Zhengjian Yang

I certify that this student has met the requirements for format contained in the University

format manual, and that this thesis is suitable for shelving in the Library and credit is to

be awarded for the thesis.

__________________________, Department Chair ___________________

Dr. Benjamin Fell Date

Department of Civil Engineering

iv
 Abstract

of
EVALUATION OF AMMONIA REMOVAL IN WASTEWATER TREATMENT

USING CONTINUOUS AERATION AND INTERMITTENT AERATION

By

Zhengjian Yang

This project focused on nitrogen contaminants and its removal process in wastewater

treatment. Moreover, this project is to evaluate nutrients removal and oxygen

consumption in the nitrification process. In this study, the experimental results reached

high ammonia removal rate (>80%) under both continuous aeration and DO= 4 mg/L,

medium ammonia removal (63%) for DO=2 mg/L, and poor ammonia removal (13.7%)

for DO=0.5 mg/L. In addition, Ammonia oxidizing bacteria (AOB) and nitrite oxidizing

bacteria (NOB) were detected in the systems. Presence of AOB and NOB under

continuous aeration and DO=4 mg/L conditions confirmed the complete nitrification

process. Future studies are encouraged to quantify the amount of AOB and NOB under

different dissolved oxygen conditions by using quantitative PCR (qPCR). Then, the HRT

should be extended to 48 hours under different dissolved oxygen. The results can be

compared with results with HRT at 24 hours. It is assumed that HRT at 48 hours will

v
result in higher ammonia removal rate than HRT at 24 hours due to a longer period for

microbial growth or decay in the system.

_______________________, Committee Chair

Dr. Amir Motlagh

_______________________
Date

vi
 ACKNOWLEDGEMENTS

This project was partially supported by Hispanic-Serving Institution’s Education Program

Grant no. 2015-38422-24058 from the USDA National Institute of Food and Agriculture.

In addition, this project was guided by Dr. Amir Motlagh from Department of Civil

Engineering, CSUS. Besides, the author would like to appreciate the local wastewater

treatment, Sacramento Regional County Sanitation District, for providing primary

effluent used in this project.

vii
 TABLE OF CONTENTS

Acknowledgements .................................................................................................... vii

List of Tables .............................................................................................................. ix

List of Figures ............................................................................................................... x

Chapter

1. INTRODUCTION……………….…………………………………………………1

2. MATERIAL AND METHODS .............................................................................. 9

3. RESULTS ............................................................................................................. 18

4. DISCUSSION ....................................................................................................... 28

5. CONCLUSION ..................................................................................................... 32

References ................................................................................................................... 33

viii
 LIST OF TABLES
Tables Page

1. List of primers and their nucleotide ………………………………… .................. 14

2. PCR conditions for different sets of primers ……………………………….…….16

ix
 LIST OF FIGURES
Figures Page

Figure 1 The schematic design of the MBBR…………………………………………..10

Figure 2 Concentrations of Ammonia ……………………………….…………………19

Figure 3 Concentrations of Nitrite…………………….. ………….……………………20

Figure 4 Concentrations of Nitrate ……………………………….…………………….21

Figure 5 Ammonia Removal ……………………………….……… . …………………21

Figure 6 Concentrations of COD ……………………………….………... ……………22

Figure 7 Removal rate of COD……………………………….……… .. ………………23

Figure 8 Ratio of VSS/TSS……………………………….……… ……………………24

Figure 9 Concentrations of BOD ……………………………….………... ……………25

Figure 10 Removal rate of BOD…………………………………………………………25

Figure 11 Intensity of DNA Bands………………………………………………………26

Figure 12 Visualization of DNA Bands…………………………………………………27

x
 1

1. Introduction

Globally, the shortage of water resources always threats human beings as well as

ecosystem. About 97% of water body is saline and since desalination is incredibly

expensive, the other 3% of the water bodies including freshwater, lakes, rivers,

groundwater, and glaciers become extremely valuable. However, many water bodies are

anthropogenically contaminated and further contamination of these environment can

significantly affect their viability.

Nitrogen is one the major sources of pollution in urban water. The US Environmental

Protection Agency (US EPA) indicates that major sources of nitrogen are fertilizers used

in agriculture, livestock wastes, cleaning products used in residential, and vehicles

emission into the atmosphere. Moreover, some industries such as coke plants, ceramic

production, strip mining, and wastewater treatment plants generate nitrogen emissions.

These sources of ammonia eventually enter into the aquatic system through nitrogen

fixation or precipitation. Nitrogen is usually found as ammonia in environment, while it

is recognized as one of the toxic chemicals, which threats the aquatic life in surface water

such as rivers, streams or lakes by over enriching the nutrients. In the most situation,

excessive Ammonia will cause large growth of algae, which is also called eutrophication

(Chislock et al., 2013). When the algae die, microorganisms in the water will consume

the available oxygen that leaves little oxygen for fish and other aquatic animals. Aguiar et

al. (2011) reported that high concentration of nitrate and nitrite resulted in the severe

eutrophication in Guanabara Bay, Brazil between 2007 and 2009. The sewage with great

amounts of organic matters were discharged directly into the bay because of absence of
 2

nearby wastewater treatment facility. Based on their measurement, the concentration of

nitrite and nitrate from the river were up to 27.63 and 10.8 µM in 2007, while 15.36 and

54.05 µM in 2008. However, the standard concentration of nitrate in the river should

range from 0.81 to 3.22 µM. Besides, Li et al. (2018) built the eutrophication model,

which was correlated to COD, dissolved inorganic nitrogen, and phosphate. As the

negative relationship between nitrogen and oxygen was detected, they demonstrated that

the increasing concentration of nitrogen would cause the phytoplankton or algae bloom

and the depletion of oxygen.

Levit (2010) indicated that small amount of ammonia excreted by the fish via urine could

be extremely toxic. Besides, ammonia could affect the central nervous system of fish as

well. The concentration of toxic ammonia (NH3) would increase in higher pH and

temperature . Also, the duration of ammonia exposure is considered as one of the crucial

factors in toxicity assessment so that US EPA set a safe value of acute ammonia

concentration of 5.9 mg-N/L as short-term exposure, while the ammonia concentration

should be less than 1.8 mg-N/L for long-term exposure. In addition, ammonia can be

toxic to other aquatic organisms such as prawns. Dutra et al. (2016) investigated

ammonia exposure to M. amazonicum as one of prawns in Amazon River. They studied

three categories of these prawns, including post-larvae, juvenile, and adults. From the

results, the safe ammonia exposure level for post-larvae was 2.114 mg-N/L. For the

juveniles, the safe exposure level was 2.165 mg-N/L, while the adults showed tolerance

at 3.659 mg-N/L as safe exposure level. Moreover, ammonia might cause the adverse

effect to the lakes by accumulation of nitrite in the water body (Chen et al., 2010).
 3

Excessive ammonia in the lake inhibits the nitrite oxidizing bacteria (NOB), causing the

accumulation of nitrite. Subsequently, the balance between growth and consumption of

the nitrite will be deteriorated and with the increasing concentration of nitrite, the lake

eutrophication is exacerbating.

As one of the sources of ammonia is wastewater, ammonia must be removed from the

wastewater before it can be discharged into water bodies. There are several physical,

chemical, and biological methods to remove ammonia, including adsorption using

activated carbon (Gonçalves et. al., 2011) or oyster shell (Shih and Chang, 2015), ion-

exchange (Krishnani et al., 2012), adsorption and oxidation (Jiang et al., 2011), and

nitrification. Due to the unique physiochemical feature of zeolite for ion exchange

sorption using seoliye is preferred and is often applied in ammonia removal. Krishnani et

al. (2012) concluded the ammonia removal rate ranges from 20% to 37% by using silver-

zeolite ion exchange method. In the contrast, Hanusova (2015) concluded adsorption

efficiency of 45.6% by using zeolite. In addition, applied microwave-enhanced

adsorption and Fenton oxidation have been practiced to remove ammonia from landfills

leachate. Microwave treatment enhanced the zeolite dispersion and electrostatic forces

while adsorbing the ammonia. Besides, other novel applications, such as oyster shell

(Shih and Chang, 2015) and new core-shell material (Si et al., 2018), have been used as

adsorption media in ammonia removal. Shih and Chang (2015) utilized oyster shell as the

media to purify the wastewater in Taipei. However, the efficiency of ammonia removal

was relatively low, which was 11.74 g/m 2.day. On the contrary, Si et al. (2018) created a

new core-shell material, which was prepared with magnesium oxide (MgO) and selenium
 4

oxide (SiO2) from the red mud. In this case, MgO reduced ammonia by adsorption in

both synthetic wastewater and real wastewater. The ammonia removal rate in the

synthetic wastewater was 76.63%, while 61.4% of ammonia was removed from the real

wastewater.

In addition to all of the above mentioned physical and chemical processes, biological

process such as nitrification has the most efficient removal rate of ammonia. The

mechanism of the nitrification process is that the ammonia is converted into nitrite

(equation 1), and then nitrite is oxidized to nitrate (equation 2).

NH4 + + 1.5O2  NO2 - + 2H+ + H2O (1)

NO2- + 0.5O2 NO3- (2)

In addition, ammonia-oxidizing bacteria (AOB) as the catalyst supports the ammonia to

be oxidized into nitrite. On the contrary, nitrite-oxidizing bacteria (NOB) stimulates the

chemical reaction from nitrite to nitrate. Although Nitrosomonas and Nitrospira are two

primary autotrophic nitrifiers, there are other bacterial species involved in nitrification

including Nitrosomonas, Comamonas, Bacillus, Nitrospira, and Pseudomonas that can be

detected in full-scale municipal wastewater treatment plant (Liu et al., 2018).

Martín-Pascual et al. (2015) constructed a hybrid moving bed biofilm reactor-membrane

bioreactor (MBBR-MBR) that reached the ammonia removal rate at maximum 98.5%

while operated on 10-hour hydraulic retention time (HRT) and 27°C. In another study,

Tang et al. (2015) proved that by adding nitrifiers into the series of batch reactors the

nitrification would significantly increase. In this study, ammonia removal increased from

0.21 mg-N/g MLVSS-h to 6.13 mg-N/g MLVSS-h with bioaugmented nitrifiers. Also,
 5

they indicated that substrate organic carbon was one of the inhibitors to the nitrification.

The lower concentration of the substrate organic carbon is in the wastewater, the lower

chance the nitrification could be inhibited. They achieved ammonia removal rate of

94.7% NH4-N after 25 days. Before the bioaugmentation, the removal rate was 75% or

lower, with effluent concentration of 28.3 mg/L of NH4-N. In the conventional

wastewater treatment, attached growth and suspended growth systems are two common

methods during the secondary treatment process to degrade some soluble organics under

aerobic conditions. For instance, activated sludge as one of the suspended growth process

settles out the floating microorganisms. In the contrast, some biological flocs are formed

and settled at the bottom of the reactor, which could be recycled for further treatment

processes. On the other hand, attached growth process involves medium to remove

microorganisms. Rotating biological contactors (RBCs) and trickling filters are two

major types. The RBC consists of rotating discs, which the microorganisms can attach

to. On the contrary, the trickling filter contains a large fixed bed media with rocks and

plastics, which can break down the organic matters. Comparing attached growth with

suspended growth, the attached growth will potentially consume fewer energy, less

bulking issues, and requires less labor (Dabi et al., 2015; Hogye, 2004). Dabi investigated

RBC attached growth and activated sludge processes by collecting wastewater samples

from a local wastewater treatment plant in India. He found that RBC saved 30% of

energy than activated sludge process. Moreover, the activated sludge process was

essential for extra management and monitoring than RBC. In addition to RBC and

trickling filter, moving bed biofilm reactor is effective in the removal of nitrogen and
 6

organic carbon. Biofilm has advantages in trapping suspended biomass in the reactor,

which provides large surface area for colonization and does not need to recycle the

biomass after the treatment (Zinatizadeh et al., 2015).

In addition to nitrifying bacterial communities, several other parameters, including pH

range, temperature, dissolved oxygen (DO), hydraulic retention time (HRT), and

chemical oxygen demand (COD) affect the efficiency of nitrification process ( Cho et

al.,2014; Shrestha et al.,2002; Zhang et al.,2014; Lin et al.,2007; Wu et al.,2016).

The optimal pH range for nitrification is between 7 and 8. Cho et al. (2014) investigated

the optimal pH range for nitrification and concluded that an optimal pH between 7.9 and

8.0 is an optimal pH for nitrifying bacteria. Besides, Shrestha et al. (2002) determined

that Nitrospira produced nitrite (NO2) effectively at an optimal pH range between 7 and

8. Cho et al. (2014) also evaluate the effects of temperature on nitrification process and

concluded that optimal temperature for nitrification was at 32.8°C. Zhang et al. (2014)

also investigated that ammonia oxidation rate significantly increased when the

temperature varied from 7.2 to 28.3°C. In addition to pH and temperature, the amount of

dissolved oxygen (DO) is affecting the ammonia removal rate. The nitrification process

occurs with certain amount of oxygen and low DO might cause relatively low ammonia

removal rate. The municipal wastewater treatment plants usually apply continuous

aeration, which generate air into the tank by diffusers all the time. This will maintain

enough amount of oxygen during the nitrification process. However, continuous aeration

has a disadvantage, which may result more energy consumption than the intermittent

aeration. Moreover, the intermittent aeration showed promising ammonia removal and a
 7

significant decrease in aeration intervals, which results in substantial reduction of energy

consumption. By reducing the amount of energy consumption, carbon footprint of

aeration process in wastewater treatment facility will be reduced. Lim et al. (2007)

performed intermittent aeration with the DO range from 0 to 7 mg/L and they discovered

that BOD and COD removal rate reached over 97% and 92%, while the ammonia

removal rate was 82% with intermittent aeration (50 minutes on aeration/70 minutes off

aeration). Furthermore, Bernet et al. (2001) proved that extremely low DO with long

solid retention time (SRT) and hydraulic retention time (HRT) may result in high

efficiency of ammonia removal. They controlled DO at 0.4 to 0.6 mg/l and operated the

reactor for 110 days with 3-day HRT. The effluent ammonia was less than 5 mg/L, while

the influent ammonia concentration was 250 mg/L. Furthermore, Wu et al. (2016)

optimized the removal of COD (97.2%) and NH4-N (98.4%) by controlling the aeration

rate at 1 L/min with operating aeration at 4 hour/day. In addition, the intermittent aeration

might significantly save energy and cost in the wastewater treatment process. For

example, Ataei (2010) compared a 24-hour aeration with an 8-hr aeration, which resulted

in a $107/day or $40,000/year difference. The continuous aeration costed $165/day, while

the intermittent aeration ended up with $58.34/day.

This study focused on the optimization of nutrients removal and oxygen consumption in

the nitrification process. Multiple experiments were involved, which included the

evaluation of ammonia removal efficiency as well as other vital parameters such as

nitrite, nitrate, and organic matter in MBBR, assessment of the effects of oxygen

concentration in ammonia removal process using continuous and intermittent aeration,

 8

and monitoring the population of microbial communities in various environmental

conditions.
 9

2. Material and Methods

2.1 A lab-scale MBBR configuration and operation

A 3-Liter lab-scale Moving Bed Bioreactor was constructed in the machine shop of

California State University-Sacramento. This plexiglass cylindrical reactor had a working

volume of 2.5 L. Meanwhile, the feed water was raw obtained from the primary effluent

at the Sacramento Regional Sanitation Distract. Two peristaltic pumps were connected

with the reactor to transfer water into and out of the system. In addition, an air stone was

connected to an air pump via tubing system and was inserted at the bottom of the reactor,

which simulated the air diffuser and provided oxygen for the nitrification process. The

temperature was controlled about 25°C, and the pH range was monitored to be between

7.1 and 7.5. Also, fluidized biofilter media (bioball) filled in 50% of the reactor

(approximately 200 of bioballs). Each filter media was about 6*7 mm. The function of

the biofilm carriers was to attach biomass in the complete mixing reactor, while microbial

communities grow on the surface of the filter media forming biofilm. This MBBR system

was initially setup as continuous aeration (with DO > 7 mg/L) with HRT 24 hr and was

operated for more than a month. During the 24-hr period, the settling time was 10 min,

while feeding and discharge periods were adjusted to 6 and 4 min, respectively. For

intermittent aeration, the aeration was limited to 90 seconds for each aeration cycle when

the DO was set to 4 mg/L, while the duration of OFF time was 10 mins between 11am

and 3:30pm, and then it was set as 1.5 hrs. Accordingly, the ON time for DO at 2 mg/L

was 30 seconds for each aeration cycle, while the OFF time lasted 30 mins from 11am to
 10

3:30pm, and followed by a 2 hrs duration. Under the condition of DO at 0.5 mg/L, the

ON time was 15 seconds for each aeration cycle, while the OFF time was 2 hours.

Figure 1: The schematic design of the MBBR.

2.2 Parameters measurement

Ammonia, nitrite, nitrate, and chemical oxygen demand (COD) were measured by

following HACH procedures( Cat.2395266). Biochemical oxygen demand (BOD), total

suspended solids (TSS), and volatile suspended solids (VSS) were measured following

the AWWA Standard Methods as explained below.

2.2.1 TSS andVSS measurement

The purpose of measuring total suspended solids from the wastewater is to indicate if

bacterial communities have experienced nutrient deficiency, because an increase in the

BOD load may cause excessive solids formation. In addition, high TSS value may also
 11

address poor aeration in the system. VSS test works as a good indicator of organic solid

concentration as well. The higher ratio of VSS and TSS means there are more microbial

communities in the system. In this case, bacteria such as ammonia oxidizing bacteria and

nitrite oxidizing bacteria are expected in the system after the nitrification process.

For measuring TSS, filters and aluminum boats were prepared and weighed on an

analytical balance. Then, the certain volume of wastewater samples from the reactor was

filtered through glass fiber filter. After that, the dirty wet filters and aluminum boat were

dried at 105° C for at least 2 hours followed by weighing them after cooling in a

desiccator.

For measuring VSS, dried filter sample and boat was placed in a muffle furnace at

500°C for 30 minutes. Then, the sample was weighed after cooling in the desiccator.

For TSS calculation, the mass differences of dirty filter and boat and the mass of clean

filter and boat was divided by the volume of filtered sample. For VSS calculation, the

mass difference of dried filter and dried filter after combustion was divided by the

volume of filtered sample.

2.2.2 BOD measurement

The purpose of the BOD test was to evaluate the amount of organic matters, which was

provided for aerobic bacterial growth as nutrient. With the increasing amount of organic

matters in the system, the more DO must is needed. BOD measures the oxygen taken up

by the bacteria during the oxidation of organic matter. There are two primary BOD

methods, respirometer and dilution method. Each method has strong correlation or linear

relation based on the study of Sibil et al. (2014). Besides, BOD 5 (five-day period test) and
 12

BODu (28-day period test) are often considered. Within a five-day period, oxidation is

60% to 70% complete, while oxidation occurs at 95% to 99% in 28 days.

In this study, a five-day period BOD test was applied by dilution method. The BOD

bottle contained the certain concentration of sample (influent and effluent from the

reactor) and dilution water were prepared. The total volume of dilution water was 2 L

along with four dilute solutions (27.5g/L of CaCl2, 0.25g/L of FeCl3, 22.5g/L of MgSO4,

and phosphate buffer). Moreover, the dilution water was aerated for more than 30

minutes prior to use. The dilution factor must be considered after the dilution water was

prepared. According to the equation, BOD = (DO i – DOf ) / f , where DOi is initial DO,

DOf is final DO, which is measured on the fifth day, and f is dilution factor (or sample

volume/300 mL). Finally, these samples were stored in the incubator for five days at

20° C.

2.2.3 Calculation of the removal rate of the parameters

The removal rate of above mentioned parameters was calculated using the equation

below:

Efficiency (%) = (Cinf – Ceff)/ Cinf * 100

Where Cinf was ammonia, nitrate nitrite COD, and BOD concentration in the influent

(mg/L), and Ceff is the corresponding concentration in the effluent.

2.3 DNA extraction and PCR for AOB and NOB

First, biomass was scrapped off from the media filters in the system in order to prepare

for DNA extraction. Then, DNA extraction was performed by following the DNeasy

PowerSoil Pro Kit procedure (QIAGEN). The complete steps include sample preparation,
 13

cell lysis, inhibitor removal, DNA binding, wash and elution. Extracted DNA with 50 μL

for each sample was stored in a freezer at –20° C for further processes. After the DNA

extraction, the NanoDrop One, UV-Vis spectrophotometer, was used to evaluate the

DNA qualities based on three major parameters, including DNA concentration, ratio of

absorption at 260 nm to absorption at 280 nm (A260/A280), and ratio of A260/A230.

Ratio of A260/A280 indicated as protein and hence, higher quality of DNA samples had

higher A260/A280 ratio. In the contrast, ratio of A260/A230 illustrated whether DNA

samples were contaminated or not and higher values demonstrated higher level of

contamination in the samples. After the DNA samples were extracted, polymerase chain

reaction (PCR) was used to amplify specific genes in the DNA samples to determine the

concentration of target genes. In this study, each PCR reaction (20 μL) included 1 μL of

DNA template, 1 μL forward primer, 1 μL reverse primer, 10 μL PCR mastermix, and 7

μL of nuclease free (NF) water. The PCR primers that were used in this study include

AOA (104F/616R), AOB (amoA1F/amoA2R), NOB1 (NSR1113f/NSR1264r),

NOB2(FGPS872f/FGPS1269r), and total bacteria (Bac518F/Bac786R) according to

previous studies (Ding et al., 2015; Harms et al., 2003). The nucleotide sequences for

these primers are listed in Table 1 below.

 14

Table 1. List of primers and their nucleotide used in this study.

Name Target Species sequence( 5' to 3') References

AOA 104F 5′-GCAGGAGACTACATMTTCTA- Ding K et

3' al.(2015)

616R 5′-GCCATCCATCTGTATGTCCA-3′

AOB amoA1F Nitrosomonas 5′-GGGGTTTCTACTGGTGGT-3'

europaea

amoA2R 5′-CCCCTCKGSAAAGCCTTCTTC-
3′ (K = G or T; S =G or C)

16S rRNA 519F 5′-CAGCMGCCGCGGTAA-3′

genes（total
archea)

727R 5′-GCTTTCRTCCCTCACCGT-3'

16S rRNA Bac518F 5′-CCAGCAGCCGCGGTAAT-3'

genes（total
bacteria)

Bac786R 5′-CTACCAGGGTATCTAATC-3′

NOB NSR1113f Nitrospira spp. 5'-CCTGCTTTCAGTTGCTACCG-3' Harms et

al.(2003)
Jingyin
Wang et
al.(2014)
 15

NSR1264r 5'-GTTTGCAGCGCTTTGTACCG-3'

NOB2 FGPS872f Winkler et

al.(2012)
Cebron et
al.(2005)

FGPS1269r

The PCR amplifications occurred in aThermal Cycler( T100, BioRad, Hercules,CA) once

the PCR mixtures were prepared. The amplification cycle included initial denaturation,

denaturation, annealing, and extension. For the amplification cycle of AOA, the initial

denaturation step was at 95°C for 2 min, followed by 40 cycles of 94°C for 40 s, 55°C for

1 min, and 72°C for 1 min. (Ding K et al. 2015). For AOB, the initial denaturation step

was at 95°C for 2 min, followed by 30 cycles of 94°C for 40 s, 55°C for 1 min, and 72°C

for 1 min. For NOB1(for detecting Nitrospira), the initial denaturation step was at 95°C

for 10 min, followed by 40 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 30 s, and

72°C for 15 min. For NOB2 (for detecting Nitrobacter), the initial denaturation step was

at 95 °C for 10 min, followed by 35 cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 1

min, and 72°C for 7 min. For total bacteria, the initial denaturation step was at 95°C for 2

min, followed by 35 cycles of 95°C for 40 s, 55°C for 40 s, 72°C for 90 s, and 72°C for

10 min. The thermal cycles for each set of primer is summarized in Table 2 below.
 16

Table 2. PCR conditions for different sets of primers used in this study.

Name PCR Thermocycle References

AOA 104F The initial denaturation step was at 95°C for 2 min, Ding K et
followed by 40 cycles of 94°C for 40 s, 55°C for 1 min, al.(2015)
and 72°C for 1 min
616R

AOB amoA1F The initial denaturation step was at 95°C for 2 min,
followed by 30 cycles of 94°C for 40 s, 55°C for 1 min,
and 72°C for 1 min.
amoA2R

16S rRNA 519F The initial denaturation step at 95 °C for 2 min, followed
genes（total by 40 cycles of 94 °C for 40 s, 55 °C for 1 min, and 72
°C for 1 min.
archea)
727R

16S rRNA Bac518F The initial denaturation step was at 95°C for 2 min,
genes（total followed by 35 cycles of 95°C for 40 s, 55°C for 40 s,
72°C for 90 s, and 72°C for 10 min.
bacteria)
Bac786R

NOB NSR1113f The initial denaturation step was at 95°C for 10 min, Harms et
followed by 40 cycles of 94°C for 30 s, 65°C for 30 s, al.(2003)
72°C for 30 s, and 72°C for 15 min. Jingyin
NSR1264r Wang et
al.(2014)

NOB2 FGPS872f The initial denaturation step was at 95 °C for 10 min, Winkler et
followed by 35 cycles of 95°C for 1 min, 50°C for 1 min, al.(2012)
72°C for 1 min, and 72°C for 7 min. Cebron et
FGPS1269r al.(2005)
 17

After PCR cycles, the original DNA samples were duplicated to millions of copies for the

target gene that would provide enough copies to visualize under UV transilluminator. The

gel electrophoresis was applied to separate the amplified gene fragments different base-

pair sequences. In this study, the 1% (w/v) gel was prepared by mixing agarose with

diluted 1x TAE buffer. After heating the agarose and TAE buffer mixture and cooling it

down, ethidium bromide (EtBr) was added so upon binding of the molecule to the DNA,

the DNA banding pattern can be visualized under UV light. PCR product and loading dye

were mixed and inserted into the well of the gel. The voltage was set at 80 V with a

running time of 40 minutes, or manually stop the system when the colored band reached

2/3 to 3/4 of the gel length. Finally, the DNA samples were visualized under the UV

illuminator ( UView, BioRad, Hercules, CA).

 18

3. Results

3.1 Nitrogen removal

The concentration of ammonia in the influent under these four conditions were various

because the various amount could witness differently in the primary effluent from the

local wastewater treatment plant. Based on figure 2, there were decreasing trend with

concentration of ammonia under all those conditions (Continuous Aeration, intermittent

aeration with DO at 4 mg/L, 2mg/L, and 0.5 mg/L, respectively). For instance, the

ammonia dropped from 71 mg/L to 13.5 mg/L in the continuous aeration. While the

effluent ammonia concentration was 7.5 mg/L, comparing with 45.5 mg/L when the DO

was controlled at 4mg/L. Meanwhile, the ammonia significantly decreased from 100

mg/L to 37 mg/L with DO at 2 mg/L. However, the concentration of ammonia decreased

only by 7 mg/L when 0.5 mg/L of DO was maintained in the system, which was 51 mg/L

in the influent. In addition, figure 4 clearly demonstrated the efficiency of ammonia

removal under these four conditions. When the DO at 4mg/L, the removal rate reached

the highest percentage of 83± 3.45%, while the DO at 0.5 mg/L provided the lowest

removal rate. Accordingly, continuous aeration and DO at 2mg/L eliminated ammonia by

81±1.3% and 63±7.29%.

 19

Concentration of Ammonia
140

120
Concentration (mg/L)

100

80

60

40

20

0
cont aeration DO=4 mg/L DO=2 mg/L DO=0.5 mg/L

inf eff

Figure 2: Concentrations of Ammonia from influent and effluent of wastewater under

four different conditions (Continuous Aeration, intermittent aeration with DO at 4 mg/L,
2mg/L, and 0.5 mg/L, respectively). Notice that all the HRT was set as a 24-hour cycle.
The concentration of nitrite was extremely low under the four conditions, showing on

figure3, which were very close (0.085mg/L for continuous aeration, 0.095 for DO at

4mg/L, 0.07 for DO at 2mg/L, and 0.08 mg/L for DO at 0.5mg/L, respectively). Besides,

there were increasing tendency under these conditions. For instance, continuous aeration

increased nitrite from 0.085 to 0.57 mg/L. Concentration of nitrite raised to 0.49 mg/L

under DO at 0.5 mg/L condition, while nitrite increased to 0.37 mg/L with DO at 2 mg/L.

However, when DO was at 4 mg/L, the concentration of nitrite barely increased to 0.1

mg/L.
 20

Concentration of Nitrite
0.7

0.6
Concentration(mg/L)

0.5

0.4

0.3

0.2

0.1

0
cont aeration DO=4 mg/L DO=2 mg/L DO=0.5 mg/L

inf eff

Figure 3: Concentrations of Nitrite from influent and effluent of wastewater under four
different conditions (Continuous Aeration, intermittent aeration with DO at 4 mg/L,
2mg/L, and 0.5 mg/L, respectively). Notice that all the HRT was set as a 24-hour cycle.
Nitrification occurred under all the conditions according to figure 4 that concentration of

nitrate experienced a certain amount of increase. Continuous aeration had the most

increase from 4.4 mg/L to 21 mg/L. While DO at 4 mg/L and DO at 2 mg/L were ranging

from 4.8 mg/L to 14.6 mg/L, and 4.4 mg/L to 18.8 mg/L, respectively. However, the DO

at 0.5 mg/L, reaching nitrate from 5 mg/L to 8 mg/L, witnessed the lowest increasing.
 21

Concentration of Nitrate
30

25
Concentration(mg/L)

20

15

10

0
cont aeration DO=4 mg/L DO=2 mg/L DO=0.5 mg/L

inf eff

Figure 4: Concentrations of Nitrate from influent and effluent of wastewater under four
different conditions (Continuous Aeration, intermittent aeration with DO at 4 mg/L,
2mg/L, and 0.5 mg/L, respectively).

Ammonia Removal
100
90
80
Removal Efficiency(%)

70
60
50
40
30
20
10
0
cont aeration DO=4 mg/L DO=2 mg/L DO=0.5 mg/L

Figure 5: Removal rate of Ammonia based on different conditions. (Continuous Aeration,

intermittent aeration with DO at 4 mg/L, 2mg/L, and 0.5 mg/L, respectively).
 22

3.2 Organics removal (COD/BOD)

There was a decreasing trend of concentration of COD based on figure 6 for all the

conditions. In this case, the influent concentration of COD was the highest in the DO at 4

mg/L, which was 1050 mg/L, and the effluent COD reduced to 420 mg/L. For DO at 2

mg/L and DO at 0.5 mg/L, the COD decreased from 350 mg/L to 110 mg/L, and 300

mg/L to 90 mg/L. On the contrary, the influent COD was measured at 130 mg/L under

the continuous aeration, while the effluent COD declined to 70 mg/L at the end.

Meanwhile, the COD removal rate were shown on figure 6 that were higher in DO at 4

mg/L, DO at 2 mg/L and DO at 0.5 mg/L with 60%, 68.6% and 70 %, respectively. The

continuous aeration had the least efficient COD removal with 46.15%.

Concentration of COD
1200

1000
Concentration(mg/L)

800

600

400

200

0
cont aeration DO=4 mg/L DO=2 mg/L DO=0.5 mg/L

inf eff

Figure 6: Concentrations of COD from influent and effluent of wastewater under four
different conditions (Continuous Aeration, intermittent aeration with DO at 4 mg/L, 2
mg/L, and 0.5 mg/L, respectively). Note that all the HRT was set as a 24-hour cycle.
 23

COD Removal
80

70

60
Removal Efficiency(%)

50

40

30

20

10

0
cont aeration DO=4 mg/L DO=2 mg/L DO=0.5 mg/L

Figure 7: Removal rate of COD based on different conditions. (Continuous Aeration,

intermittent aeration with DO at 4 mg/L, 2mg/L, and 0.5 mg/L, respectively).
3.3 Ratio of VSS and TSS

Figure 8 illustrated the f-ratio of VSS/TSS under the four conditions. The highest ratio

was 84.22% when the DO at 2 mg/L. In the contrast, other conditions all experienced

ratio over 70%, which were 76.2% for continuous aeration, 76.4% for DO at 4mg/L, and

73.3% for DO at 0.5 mg/L.

 24

Ratio of VSS and TSS

86.00%
84.00%
82.00%
80.00%
78.00%
f(%)

76.00%
74.00%
72.00%
70.00%
68.00%
66.00%

Continuous Aeration DO=4mg/L DO=2mg/L DO=0.5mg/L

Figure 8: Ratio of VSS/TSS under different conditions ( Continuous Aeration, intermittent

aeration with DO at 4 mg/L, 2mg/L, and 0.5 mg/L, respectively).
3.4 BOD measurement

The BOD concentration, based on figure 9, decreased significantly under continuous

aeration, DO at 4 mg/L, and DO at 0.5 mg/L. However, the BOD was only reducing from

53 mg/L to 24.2 mg/L when DO was 2 mg/L. Meanwhile, figure 9 illustrated that the DO

at 2 mg/L had the lowest removal efficiency at 54.4%. On the contrary, the removal rate

was much higher under other conditions, which were 89% for continuous aeration, 96%

for DO at 4 mg/L, and 84.67% for DO at 0.5 mg/L.

 25

Concentration of BOD
350

300

250
Concentration(%)

200

150

100

50

0
cont. aeration DO=4mg/L DO=2mg/L DO=0.5mg/L

inf(mg/L) eff(mg/L)

Figure 9: Concentrations of BOD from influent and effluent of wastewater under four
different conditions (Continuous Aeration, intermittent aeration with DO at 4 mg/L,
2mg/L, and 0.5 mg/L, respectively). Notice that all the HRT was set as a 24-hour cycle.

BOD Removal
120

100
Removal Efficiency(%)

80

60

40

20

0
cont. aeration DO=4mg/L DO=2mg/L DO=0.5mg/L

Figure 10: Removal rate of BOD based on different conditions. (Continuous Aeration,
intermittent aeration with DO at 4 mg/L, 2 mg/L, and 0.5 mg/L, respectively).
 26

3.5 Nitrifying bacteria characteristics

The DNA bands represented AOBs and NOB1(nitrospira) from the left to the right on

figure 11. Then, figure 10 was established to identify the intensity of each band. The

strongest bands were both showing related to AOB under continuous aeration and DO=4

mg/L conditions, following with the medium band in DO=2mg/L and faint band in

DO=0.5mg/L. Accordingly, nitrospira communities were detected in continuous aeration

and DO=4 mg/L. Nitrobacter and AOA were missing in the bacterial community.

Intensity rating of amplified DNA

3
DNA band intensity

0
DO=4 mg/L

DO=2 mg/L

DO=0.5 mg/L

DO=4 mg/L

DO=2 mg/L

DO=0.5 mg/L

DO=4 mg/L

DO=2 mg/L

DO=0.5 mg/L

DO=4 mg/L

DO=2 mg/L

DO=0.5 mg/L
cont aeration

cont aeration

cont aeration

cont aeration

amoA(AOB) NOB-1(nitrospira) NOB-2(nitrobacter) AOA

Figure 11: The intensity of DNA bands (AOB, NOB1,NOB2, and AOA).
 27

Figure 12: The visualization of DNA bands under the UV illuminator.

 28

4. Discussion

4.1 Ammonia removal

Based on figure 1 and figure 4, continuous aeration and intermittent aeration with DO=4

mg/L had relatively high removal rate (81% and 83%). Moreover, DO =4 mg/L had even

higher removal rate than continuous aeration. A reasonable assumption that the system

might contain more AOB among the microbial communities than the continuous aeration.

In addition, figure 2 and figure 3 illustrated the formation of nitrite and nitrate during the

nitrification processes. Continuous aeration conducted the most conversion from

ammonia to nitrite and nitrate. While DO= 0.5 mg/L produced the least nitrate due to the

least oxygen. From figure 5 to figure 8, the concentration changes of the organic matter

contents (COD and BOD) were displayed. COD was to measure most chemicals in the

water that can be oxidized. While BOD was to measure the amount of food (or organic

carbons) that bacteria can oxidize. Moreover, COD and BOD consumed oxygen, which

reduced the amounts of oxygen for bacteria. In this case, these organic matters were

recognized as inhibitors for the nitrification. Among these conditions, the COD was

mostly removed from 60% to 70%, and BOD was eliminated above 80%. Hence, this

indicated the nitrification processes were under a decent environment.

Based on the results of figure 1 and figure 4, the continuous aeration (with DO larger

than 8 mg/L, 24-hour HRT, and 10-day SRT) provided the relatively high removal rate at

80% as well as BOD removal rate at 89%. Besides, the temperature was controlled at 25

degree Celsius, and pH was maintained at 7.5. Comparing with other MBBR, Zhang et al.

(2016) built their MBBR with cubic-shaped polyurethane sponges as biofilms. They
 29

compared with three filling fraction of the sponge carries (10%, 20%, and 30% filling

with the reactor). They obtained the highest ammonia removal rate at 98% by filling 20%

of the reactor with an average 12-hr HRT. Meanwhile, the DO concentration was

controlled from 5 to 6.5 mg/L, which could be assumed as continuous aeration. Their pH

was adjusted at 7. In addition, Wang et al. (2006) filled biofilm carriers about 50% of

their MBBR under room temperature. Besides, their pH ranged from 6.2 to 7.5. The

influent ammonia was average at 54 mg/L, while COD was from 145 to 432 mg/L. Their

purpose was to figure out the most removal efficiency under different DO concentration

(1, 2, 4, and 6 mg/L) with HRT of 6 hours. Based on the results,the continuous aeration

(or DO at 6 mg/L) provided the maximum ammonia removal at 99.2%, while COD and

BOD were eliminated by 78.7% and 77.1%, respectively. Although their reactor had

much less HRT than our reactor, they still witnessed a higher ammonia removal rate. A

reasonable guess was that high concentration of AOB involved in their system. However,

they did not provide any data related to the AOB. Lim et al. (2012) also compared with

filling ratio of biofilms in their reactor. They indicated that about 30% of filling would

remove total nitrogen completely. In contrast, they initialized the removal rate of 57% by

filling 8% of the reactor. Hence, the number of biofilms filled in the reactor might be

correlated to the removal efficiency.

For intermittent aeration, DO at 4 mg/L had the highest ammonia removal efficiency at

85.4%, which was significantly higher than low DO conditions (2 mg/L and 0.5 mg/L).

Lim et al. (2007) mentioned that the higher total nitrogen removal efficiency occurred

with the longer aeration off time. Their results indicated that aeration with 50min on /
 30

70min off reduced 97% of ammonia and 87% total nitrogen. This study with DO at 0.5

mg/L had the longest off time, but it produced lowest ammonia removal rate at only

13.7%. We believe that Lim et al. had higher DO (maximum at 7 mg/L) as well as longer

time to pump the oxygen into their system. Hence, our results were opposite from their

report, which removal rate was not correlated to on/off time. Besides, this situation was

reasonable that efficient nitrification depends on the amount of oxygen dissolved in the

water. The higher concentration of oxygen, the more ammonia would be converted to

nitrite and nitrate. In addition, although the least ammonia removal efficiency was under

DO at 0.5 mg/L, we suggest the efficiency would increase if the system had longer

enough SRT and HRT. Bernet et al. (2001) studied the nitrification under low DO at 0.5

mg/L with an average HRT of 3 days and SRT of 170 days. They operated the high DO

at the first 15 days, and then they switched the reactor to low DO condition. Although the

ammonia oxidizer declined after the few days when the system was switched to low DO,

the growth of Nitrosomonas and other ammonia oxidizers recovered afterwards. The

ammonia was reduced to 5 mg/L after 50 days, and the removal rate reached over 90%

after 110 days.

4.2 Nitrification performance

In this study, amoA as AOB, nitrospira as NOB were detected in the systems. For

instance, figure 10 and figure 11 indicated that strong bands of amoA occurred in both

continuous aeration and DO=4 mg/L. Meanwhile, medium bands showed in DO=2 mg/L,

and faint bands was in DO=0.5 mg/L. In contrast, nitrospira were witnessed in

continuous aeration and DO=4 mg/L. Analyzing with ammonia removal rate under these
 31

conditions, DO=2 mg/L and DO=0.5mg/L contained fewer AOB so that ammonia was

less oxidized than continuous aeration and DO=4mg/L. However, the intensity of bands

was awkward to distinguish the amount difference of AOBs between continuous aeration

and DO=4mg/L. In addition, the DNA bands of nitrospira could indicate that the

strongest and medium bands in continuous aeration and DO=4 mg/L, which emphasized

the nitrification occurred mostly in these two situations. Now that current experiment

results could not demonstrate why DO=2mg/L experienced better nitrification

performance than the continuous aeration. The system assumed to involve greater

amounts of active nitrospira than DO=4 mg/L. In addition, this study did not detect any

AOA, although our experiment used the same AOA and AOB primers as Ding et

al.,(2015). They indicated that AOA responded more sensitive than AOB in the

nitrification due to the positive correlation between AOA and total nitrogen. Meanwhile,

the qPCR determined that the amounts of archaeal amoA genes were significant more

than the bacterial amoA genes. Besides, nitrosomonas occupied 6% to 23% of AOB,

while nitrosospira dominated the AOB community as 77% to 93%. On the contrary,

nitrospira was dominant in NOB but significantly less than AOB, similar to Harms et

al.,(2003). Their study reported 8.6% of nitrospira was detected among total bacterial

population. Furthermore, the AOB also outnumbered by 2 magnitude than NOB in the

research of Wang et al.,(2014). They discovered that both AOB and NOB increased with

the increasing of DO, which was illustrated on figure 11 in this study as well. To obtain

more accurate experiment results, further investigation will be essential to estimate the

quantity of AOBs and NOBs under all the conditions by using qPCR method.
 32

5. Conclusion

In this study, the experimental results reached high ammonia removal rate (>80%) under

both continuous aeration and intermittent DO=4 mg/L, medium ammonia removal (63%)

for DO=2 mg/L, and poor ammonia removal (13.7%) for DO=0.5 mg/L. In addition,

Ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were detected

in the systems. Presence of AOB and NOB under continuous aeration and DO=4 mg/L

conditions confirmed the complete nitrification process.

Future studies are encouraged to quantify the amount of AOB and NOB under different

dissolved oxygen conditions by using quantitative PCR (qPCR). The HRT should be

extended to 48 hours under different dissolved oxygen by comparing all the results with

HRT at 24 hours. There is an assumption that HRT at 48 hours will result in higher

ammonia removal rate than HRT at 24 hours due to a longer period for microbial growth

or decay in the system.

 33

References

Ataei, A. (2010). Wastewater treatment: energy-conservation opportunities: consider

these options to improve energy efficiency and reduce the cost of treating

wastewater. Chemical Engineering, 117(1), 34-42.

Bernet, N., Dangcong, P., Delgenès, J. P., & Moletta, R. (2001). Nitrification at low

oxygen concentration in biofilm reactor. Journal of environmental engineering,

127(3), 266-271.

Chen, G., Cao, X., Song, C., & Zhou, Y. (2010). Adverse effects of ammonia on

nitrification process: the case of Chinese shallow freshwater lakes. Water, Air, &

Soil Pollution, 210(1-4), 297-306.

Chislock, M. F., Doster, E., Zitomer, R. A., & Wilson, A. E. (2013). Eutrophication:

causes, consequences, and controls in aquatic ecosystems. Nature Education

Knowledge, 4(4), 10.

Cho, K. H., Kim, J. O., Kang, S., Park, H., Kim, S., & Kim, Y. M. (2014). Achieving

enhanced nitrification in communities of nitrifying bacteria in full-scale

wastewater treatment plants via optimal temperature and pH. Separation and

Purification Technology, 132, 697-703.

Dabi, N. (2015). Comparison of suspended growth and attached growth wastewater

treatment process: a case study of wastewater treatment plant at MNIT, Jaipur,

Rajasthan, India. Europ. J. of Adv. in Eng. and Techn, 2(2), 102-105.

 34

De Carvalho Aguiar, V. M., Neto, J. A. B., & Rangel, C. M. (2011). Eutrophication and

hypoxia in four streams discharging in Guanabara Bay, RJ, Brazil, a case study.

Marine pollution bulletin, 62(8), 1915-1919.

Ding, K., Wen, X., Li, Y., Shen, B., & Zhang, B. (2015). Ammonia-oxidizing archaea

versus bacteria in two soil aquifer treatment systems. Applied microbiology and

biotechnology, 99(3), 1337-1347.

Dutra, F. M., Forneck, S. C., Brazão, C. C., Freire, C. A., & Ballester, E. L. C. (2016).

Acute toxicity of ammonia to various life stages of the Amazon river prawn,

Macrobrachium amazonicum, Heller, 1862. Aquaculture, 453, 104-109.

Gonçalves, M., Sánchez-García, L., Oliveira Jardim, E. D., Silvestre-Albero, J., &

Rodríguez-Reinoso, F. (2011). Ammonia removal using activated carbons: effect of the

surface chemistry in dry and moist conditions. Environmental science &

technology, 45(24), 10605-10610.

Hanusová, A. (2014). The Use of Filtration Materials to Remove Ammonia from

Water/Využitie Filtračných Materiálov Na Odstraňovanie Amoniaku Z Vody.

GeoScience Engineering, 60(4), 29-38.

Harms, G., Layton, A. C., Dionisi, H. M., Gregory, I. R., Garrett, V. M., Hawkins, S. A.,

... & Sayler, G. S. (2003). Real-time PCR quantification of nitrifying bacteria in a

municipal wastewater treatment plant. Environmental science & technology,

37(2), 343-351.
 35

Hogye, S. (2004). The attached growth process an old technology takes new forms. J.

Pipe Line, 15(1), 1-8.

Jiang, B., Meng, F., Ma, T., Jiang, L., Liu, X., & He, R. (2011, June). Research on m

method of adsorption and oxidation enhanced by microwave treating urban

landfill leachate. In 2011 International Conference on Remote Sensing,

Environment and Transportation Engineering (pp. 6233-6237). IEEE.

Krishnani, K. K., Zhang, Y., Xiong, L., Yan, Y., Boopathy, R., & Mulchandani, A.

(2012). Bactericidal and ammonia removal activity of silver ion-exchanged zeolite.

Bioresource technology, 117, 86-91.

Levit, S. M. (2010). A literature review of effects of ammonia on fish. Center for Science

in Public Participation Bozeman, Montana (2010). ttps://www.

conservationgateway. org/ConservationByGeography/North

America/UnitedStates/alaska/sw/cpa/Documents/L2010ALR122010. pdf.

Li, Q., Wang, C., Jiang, J., & Chen, S. (2018). Spatiotemporal Changes of Nutrients and

Eutrophication in a Semi-Enclosed Bay, Southeast China.

Lim, B. S., Choi, B. C., Yu, S. W., & Lee, C. G. (2007). Effects of operational

parameters on aeration on/off time in an intermittent aeration membrane

bioreactor. Desalination, 202(1-3), 77-82.

 36

Lim, J. W., Lim, P. E., & Seng, C. E. (2012). Enhancement of nitrogen removal in

moving bed sequencing batch reactor with intermittent aeration during REACT

period. Chemical Engineering Journal, 197, 199-203.

Liu, F., Hu, X., Zhao, X., Guo, H., Zhao, Y., & Jiang, B. (2018). Rapid nitrification

process upgrade coupled with succession of the microbial community in a full-

scale municipal wastewater treatment plant (WWTP). Bioresource technology,

249, 1062-1065.

Martín-Pascual, J., Reboleiro-Rivas, P., López-López, C., Leyva-Díaz, J. C., Jover, M.,

Muñio, M. M., ... & Poyatos, J. M. (2015). Effect of the filling ratio, MLSS, hydraulic

retention time, and temperature on the behavior of the hybrid biomass in a hybrid

moving bed membrane bioreactor plant to treat urban wastewater. Journal of

Environmental Engineering, 141(7), 04015007

Shih, P. K., & Chang, W. L. (2015). The effect of water purification by oyster shell

contact bed. Ecological Engineering, 77, 382-390.

Shrestha, N. K., Hadano, S., Kamachi, T., & Okura, I. (2002). Dinitrogen production

from ammonia by Nitrosomonas europaea. Applied Catalysis A: General, 237(1-

2), 33-39.

Si, Q., Zhu, Q., & Xing, Z. (2018). Simultaneous removal of nitrogen and phosphorus by

magnesium-modified calcium silicate core-shell material in water. Ecotoxicology

and environmental safety, 163, 656-664.

 37

Sibil, R., Berkun, M., & Bekiroglu, S. (2014). The comparison of different mathematical

methods to determine the BOD parameters, a new developed method and impacts

of these parameters variations on the design of WWTPs. Applied Mathematical

Modelling, 38(2), 641-658.

Tang, H. L., & Chen, H. (2015). Nitrification at full-scale municipal wastewater

treatment plants: Evaluation of inhibition and bioaugmentation of nitrifiers.

Bioresource technology, 190, 76-81.

Wang, J., Zhang, C., & Rong, H. (2014). Analysis and succession of nitrifying bacteria

community structure in sequencing biofilm batch reactor. Applied microbiology

and biotechnology, 98(10), 4581-4587.

Wang, X. J., Xia, S. Q., Chen, L., Zhao, J. F., Renault, N. J., & Chovelon, J. M. (2006).

Nutrients removal from municipal wastewater by chemical precipitation in a

moving bed biofilm reactor. Process Biochemistry, 41(4), 824-828.

Wu, H., Fan, J., Zhang, J., Ngo, H. H., Guo, W., Hu, Z., & Lv, J. (2016). Optimization of

organics and nitrogen removal in intermittently aerated vertical flow constructed

wetlands: effects of aeration time and aeration rate. International Biodeterioration

&Biodegradation, 113, 139-145.

Zhang, S., Wang, Y., He, W., Wu, M., Xing, M., Yang, J., ... & Pan, M. (2014). Impacts

of temperature and nitrifying community on nitrification kinetics in a moving-bed

biofilm reactor treating polluted raw water. Chemical Engineering Journal, 236,

242-250.
 38

Zhang, X., Chen, X., Zhang, C., Wen, H., Guo, W., & Ngo, H. H. (2016). Effect of filling

fraction on the performance of sponge-based moving bed biofilm reactor. B

Bioresource technology, 219, 762-767.

Zinatizadeh, A. A. L., & Ghaytooli, E. (2015). Simultaneous nitrogen and carbon

removal from wastewater at different operating conditions in a moving bed

biofilm reactor (MBBR): process modeling and optimization. Journal of the

Taiwan Institute of Chemical Engineers, 53, 98-111.