Thesis _Yue Liu

Abstract
Filamentous bulking sludge due to excessive growth of filamentous bacteria is a serious operational
problem in activated sludge systems. The addition of chemicals is one of widespread ways to
control filamentous sludge bulking problem. Instantaneous improvement of the settling of bulking
filamentous activated sludge can be achieved by one-time addition of chemicals. Long-term
improvement relies on repeated additions since these additives have no adverse effects on the
filaments. This study demonstrated that an alternative new chemicals, nano zero-valent iron (NZVI)
can exhibit a much stronger adverse effect on the filaments, which could also improve sludge
settleability. In two simulative activated sludge treatment systems, several conditions were
conducted by adding NZVI to the tank #2 at the final concentration of 37.5 mg Fe/L (One-time
dosing), 75 mg Fe/L (Two-consecutive-time dosing), 112.5 mg Fe/L (Three- consecutive-time
dosing), respectively. In addition, tank 1 was chosen as a control. The side effect of the use of
NZVI depended on bulking conditions and biomass concentration. In the system with sludge
bulking and significantly sludge loss, the average biomass concentration reduced to 1500 mg/L.
After dosing NZVI, the systems was caused a significant increase in effluent COD, and NH4
+
-N
and NO2
-
-N concentrations. On the other hand, with the early stages of bulking and the biomass
concentration of 2000 mg/L, the effluent water quality and overall bioreactor performance were
slightly affected for several days. The results suggest that NZVI dosing is a promising new method
for filamentous sludge bulking control.
Keywords: filamentous sludge bulking, nano zero-valent iron (NZVI), activated sludge

摘要
丝状菌的过度生长而导致的丝状菌污泥膨胀对于活性污泥装置是一个严重的操作问题。
应对丝状菌污泥膨胀最常见的方法是添加化学物质。我们可通过添加化学物质迅速提升污
泥的沉降性能，而由于所添加化学物质本身对丝状菌无害，我们只能通过重复添加来获得
长期抑制效果。本实验通过两个模拟的活性污泥处理系统发现，新型替代品——纳米零价
铁可以在提升污泥沉降性的同时有效抑制丝状菌生长。实验中，Tank #1 作为对照组，
Tank #2 则分别在不同阶段投加不同浓度的纳米铁，浓度为 37.5 mg Fe/L（第一次投加），
75 mg Fe/L（第二次连续投加），112.5 mg Fe/L（第三次投加）。我们还发现纳米铁所产
生的副作用取决于膨胀条件和菌种浓度。实验结果表明，由于污泥膨胀和明显的污泥损失
导致了污泥平均浓度降至 1500 mg/L。在投加了纳米铁后，系统中出水 COD，氨氮和亚硝
氮浓度均有上升。在另一方面，在污泥膨胀初期，污泥浓度达到 2000 mg/L 时，出水水质
和整体生物反应器性能均未受到明显影响。综上，纳米零价铁将会是治理丝状菌污泥膨胀
的一种有发展前景的新型试剂。
关键词：丝状菌污泥膨胀，纳米零价铁，活性污泥

TABLE OF CONTENTS
1. INTRODUCTION ..................................................................................................................1
2. LITERATURE REVIEW ......................................................................................................6
2.1 Activated sludge system.............................................................................................................6
2.1.1 Definition and purpose............................................................................................................6
2.1.2 Process description..................................................................................................................7
2.1.3 Factors affecting performance.................................................................................................8
2.2 Sludge bulking .........................................................................................................................10
2.2.1 Definition ..............................................................................................................................10
2.2.2 Current theories to explain bulking sludge ...........................................................................10
2.2.3 Influence factors....................................................................................................................12
2.2.4 Morphological relationship between filaments and flocs .....................................................15
2.2.5 Filamentous sludge bulking control......................................................................................15
2.3 NZVI in wastewater treatment.................................................................................................18
2.3.1 Applications ..........................................................................................................................18
2.3.2 NZVI synthesis......................................................................................................................18
2.3.3 Feasibility and advantages of NZVI for sludge bulking control...........................................20
2.4 Research Objectives.................................................................................................................21
3. MATERIALS AND METHODS .........................................................................................21
3.1 Nano zero-valent iron synthesis and characterization..............................................................21
3.2 CSTR (bioreactor) setup and operation....................................................................................23
3.3 Feedstock for CSTR systems ...................................................................................................26

3.4 Main methods involved in the research ...................................................................................27
3.4.1 Effect of NZVI dosing on nitrifying activity ........................................................................27
3.4.2 Microscopic, chemical and water quality analysis................................................................28
3.4.3 Filamentous bacterial DNA extraction for q-PCR analysis ..................................................29
3.5 Batch study...............................................................................................................................30
3.5.1 Objective ...............................................................................................................................30
3.5.2 Material and methods............................................................................................................30
3.6 Experimental implement ..........................................................................................................33
4. RESULTS AND DISCUSSION ...........................................................................................34
4.1 Sludge bulking and bioreactor performance ............................................................................34
4.2 Bioreactor performance and benefits associated with NZVI dosing .......................................40
4.3 Impact of sludge bulking and NZVI dosing on nitrifying bacterial activity............................44
5. CONCLUSIONS ...................................................................................................................45
6. FUTURE STUDY .................................................................................................................45
6.1 Change of some conditions before dosing ...............................................................................45
6.2 Bioreactor performance recovery.............................................................................................46
6.3 Sludge bulking associated with long changeable SRT operation ............................................46
6.4 Impact of sludge bulking and NZVI dosing on nitrifying bacterial population.......................46
6.5 Identification of filamentous bacteria before and after NZVI dosing......................................47
ACKNOWLEDGEMENTS.........................................................................................................48
DEDICATION..............................................................................................................................49
7. REFERENCES......................................................................................................................50

LIST OF ABBREVIATIONS
NZVI Nano Zero-Valent Iron
WWTPs Wastewater Treatment Plants
BNR Biological Nutrients Removal
CTAB Cetyltrimethylammonium Bromide
F/M Food-to-Microorganisms
DO Dissolved Oxygen
COD Chemical Oxygen Demand
SVI Sludge Volume Index
SRT Solids Retention Time
HRT Hydraulic Retention Time
MLSS Mixed Liquor Suspended Solids
VSS Volatile Suspended Solids
SOUR Specific Oxygen Uptake Rate
CSTR Continuous Stirred Tank Reactor
q-PCR Quantitative Polymerase Chain Reaction
TEM Transmission Electron Microscopy
AOB Ammonia-Oxidizing Bacteria
NOB Nitrite-Oxidizing Bacteria
T-RFLP Terminal Restriction Fragment Length Polymorphism

Filamentous sludge bulking control by nano zero-valent iron in activated sludge treatment systems 1
1. INTRODUCTION
Activated sludge (Figure 1.1) is a most commonly used process for treating sewage and industrial
wastewaters using air and a biological floc composed of bacteria and protozoa in the biological
wastewater treatment plants (Eikelboom et al. 1998). During the treatment, the activated sludge
can be applied to obtain the following purposes: 1) oxidizing carbonaceous biological matter; 2)
oxidizing nitrogenous matter, main ammonium and nitrogen in biological matter; 3) removing
phosphates; 4) generating a biological floc that is easy to settle; 5) generating a liquor that is low
in dissolved or suspended. According to the structure of the system, it consists of two stages, which
are biochemical stage (aeration tank) and physical stage (final clarifier). The biochemical stage is
the main component of the whole system. In the stage, organic carbon, ammonium and phosphate
are efficiently removed from the wastewater by the microorganisms in the aeration tank. There
exist a very large quantities of species of viruses, bacteria, protozoa, fungi, metazoan and algae,
which can be used to treat the wastewater (Martins et al. 2004). The performance of this process
relies a lot on a good solid-liquid separation between the treated water and the sludge in the final
clarifier, which leads to a good effluent quality from the activated sludge process (Clauss et al.
1999).
Sludge bulking is a terminology used to describe a condition occurs when the sludge fails to
separate out in the sedimentation tanks. For the different causes of the problem (foaming, pin-point
flocs, viscous bulking and filamentous bulking), the performance of this important process can be
worse. The excessive growth of filamentous bacteria in the sludge, which is referred to as
“filamentous bulking”, is the most common cause of poor settling problem (Krhutková et al. 2002).

Figure 1.1 A generalized, schematic of an activated sludge process (From website: http://en.wikipedia.org/wiki/Activ
ated_sludge).
The rest is referred to as “non-filamentous bulking”, which includes forming, pin-point flocs,
viscous bulking. Since the most common cause is filamentous bacteria, a lot of research have been
studied about it. Some literatures demonstrated that the filamentous bacteria regularly do not
represent the dominant metabolic bacterial group in the treatment plant, but still cause bulking
sludge (Kappeler and Gujer 1994). It results in looser and less settleable sludge flocs, which cause
sludge loss from the clarifiers and deterioration of effluent water quality (Guo et al. 2012). Despite
much related research bulking sludge seems to be a continuous problem in operational wastewater
treatment plants. More than 50% of the wastewater treatment plants (WWTPs) in the U.S. are
reported to have sludge bulking problems (Lemmer 2003). Currently, the documents recorded
about the mechanisms of sludge bulking and the plant operation under which bulking sludge occurs
Treated waterRaw water
Aeration Tank
Air
Clarifier-Settler
To Sludge Treatment
Recycle Sludge
Water Sludge

are limited. This is the biological system, it is not easy enough to explain the whole change and
development of the process. There is no single mechanism that can fully explain the sludge bulking
problems (Lou and Zhao 2012). One reason for not figuring out a good general solution to bulking
sludge possible be the absence of a consensus on the exact level at which the problem should be
approached. The main solution is to identify the specific filamentous bacteria in the bulking sludge
by applying q-PCR (Quantitative Polymerase Chain Reaction) work. Based on the results, the
species of filamentous could be identified and specific chemicals are used to selectively kill the
filamentous bacteria (Eikelboom 2000). Another approach is the recognition that the general
characteristic is the cell morphology. Since realizing how the conditions could affect the bacteria
lead to a general solution. In practice, the causes for filaments growth in activated sludge treatment
are complex and include factors such as low food-to-microorganisms (F/M), long solids retention
time (SRT), low dissolved oxygen (DO) or low nutrients (Jenkins et al. 2004, Wanner 1994). Type
021N, Type 1701, Microthrix parvicella, Thiothrix spp, Gordonia spp., were found to be
responsible for most of the filamentous sludge bulking problems.
Practical control methods for filamentous sludge bulking include specific and non-specific method
(Martins et al. 2004). Specific methods are intended to eliminate the causes favorable for
filamentous growth. Since the growth of filamentous bacteria can be encouraged easily under a
broad range of environmental conditions, it is difficult to find a unique environment that
consistently favors the growth of floc-forming bacteria while selectively kill filamentous bacteria
(Guo et al. 2010). Therefore, non-specific methods are more commonly employed by adding
substances directly to the sludge to readily improve the settleability (Guo et al. 2012).

These adding substances are diverse and have different modes of action. Three main groups can be
distinguished as follows. The biocides, such as chlorine and hydrogen peroxide, are practically
applied. This approach is based on the fact that filaments protrude from the flocs are susceptible to
toxicant exposure, which most of floc-forming bacteria are embedded inside the flocs protected
from exposure to toxicants. In other words, the use of biocides aims at a selective killing of the
filamentous bacteria impeding the sedimentation improvement. Chlorination is the most widely
applied sludge bulking control substance due to its low cost and easily obtainable (Bitton 2010).
However, this solution hardly yield immediate sedimentation improvement, but in turn, represents
a longer-term solution. Furthermore, chlorination has adverse effect on wastewater treatment
performance by deflocculating activated sludge leading to poor effluent water quality (Ramírez et
al. 2000, Wimmer and Love 2004). On the other hand, there exist chlorine-resistant filamentous
bacteria in the activated sludge (Séka et al. 2001). Other types of toxicants such as
cetyltrimethylammonium bromide (CTAB) are too costly to consume (Guo et al. 2012). The
ballasting agents (mostly the talc based) are used to weight the sludge, and further reinforcing the
flocs structure (Clauss et al. 1999). This approach is characterized by an immediate sludge
sedimentation improvement. They have no adverse effects on the filaments causing the bulking
compared with the flocculating agents. The coagulating and flocculating agents, represented by
synthetic polymers, aims at overcoming the bridging or diffuse floc structure associated with excess
filamentous microorganisms’ growth, which can also be used to improve sludge sedimentation
(Jenkins et al. 2004). However, coagulation and flocculation could not kill filamentous bacteria
(Bitton 2010).

Obviously there is a need to explore a novel method to control sludge bulking problems. To do this
end, a novel additive can be formulated based on lab scale. Nanomaterials can be the new type of
materials that may have beneficial uses in wastewater treatment. They are highly reactive and often
differ in many aspects of characteristics compared to their bulk counterparts (Maynard et al. 2006).
By virtue of their size, nanomaterials have been shown to possess distinctive chemical, catalytic,
electronic, magnetic, mechanical and optical properties (Jortner and Rao 2002). For the past several
years, nanoscale metallic iron (NZVI), has been investigated as a new tool for the treatment of
contaminated water (Crane and Scott 2012). It is one of the most commonly used and studied
engineered nanoparticles due to its widespread applications (Elliott et al. 2009, Lee et al. 2008).
The technology has reached commercial status in many countries worldwide. At nanoscale, the
specific surface area of zero valent iron increases dramatically and hence the surface reactivity of
nanoscaled iron particles is more effective remediation than meshed iron powder (Yuvakkumar et
al. 2011). Furthermore, NZVI has been evaluated in wastewater treatment for nitrogen removal
through chemical reduction of nitrate (Shin and Cha 2008) and phosphate removal through
chemical precipitation (Chang et al. 2008). It was also reported that the associated release of Fe2+
due to oxidative dissolution of NZVI helps sludge flocculation and settling (Wilén et al. 2004). In
addition, NZVI was evaluated to be a highly selective agent (Marsalek et al. 2012a). Since
filamentous bacteria and NZVI have high surface/volume ratios, it is hypothesized that filamentous
bacteria are more susceptible to NZVI exposure than floc-forming bacteria, thus leading to
selectively remove filamentous bacteria.

2. LITERATURE REVIEW
2.1 Activated sludge system
2.1.1 Definition and purpose
The activated sludge process was developed in England in 1914 (Ardern and Lockett 1914). Since
then, the activated sludge process has grown in popularity until today it is the most widely used
biological wastewater treatment process. It is an aerobic suspended growth process that is widely
applied to treat sewage and industrial wastewaters using air and a biological floc composed of
bacteria and protozoa. The microorganisms involved in the system are grown in a variety of
bioreactor configurations for the purpose of removing soluble organic matter. It is widely accepted
to be a reliable and flexible process capable of producing a high quality effluent. A clear effluent
low in suspended solids is produced due to the flocculent and sedimentation nature of the biomass.
Thus, activated sludge is probably the most versatile of the biological treatment processes. During
the process, nitrification and stabilization of insoluble organic matter can also be highly achieved
by operation at an appropriate long solids retention time (SRT). Based on the past practice, the
process is controllable and its operation can be adjusted in response to a wide range of conditions.
On the other hand, the system could relatively resistant to hydraulic loading variations. The main
reason for not appropriate is a result of its controllability. The conditions are variable and relatively
complicated (Grady Jr et al. 2011).
Activated sludge is a biological contact process where bacteria, fungi, protozoa and some small
organisms. It is obvious that the bacteria are the most important group of microorganisms for they
are the ones responsible for the structural and functional activity of the activated flocs. There exist

many types of bacteria that make up the whole activated sludge system. The predominant type of
bacteria should be determined by the components in the wastewater, the operation conditions of
the plant, and the environmental conditions present for the organisms in the process. However,
fungi are relatively rarely in the system. Once they present, most of the fungi tend to be the
filamentous forms which prevent good floc formation and therefore make negative effect on the
performance of the plant. Several factors that can lead to stimulate fungi growths. Low dissolved
oxygen concentrations, nutrient deficiencies, and unusual organic compounds are the main
conditions that could cause fungi to growth significantly.
As for purposes and objectives, they can be described as follows. The system can oxidize
carbonaceous biological matter and nitrogenous matter (mainly referred to nitrification). In
addition, removing phosphates and pushing off entrained gases are also included in the process.
The last two purposes are to generate biological flocs that is easy to settle and a liquor that is low
in dissolved or suspended material (Grady Jr et al. 2011).
2.1.2 Process description
The activated sludge must be kept in suspension during the contact with the wastewater. Therefore
the process (Figure 1.1) consists of the following steps:
(1) Mixing the activated sludge with the wastewater to be treated, referred to mixed liquor,
which occurs in the aeration basin.
(2) Aeration and agitation of the mixed liquor for the required length of time.
(3) Separation of the activated sludge from the mixed liquor, in the final clarification process,
which occurs in the clarifier.

(4) Return the proper amount of activated sludge for mixture with the wastewater to maintain
the mixed liquor suspended solids (MLSS) concentration.
(5) Disposal of the excess activated sludge.
Aeration basins are typically open tanks containing equipment to transfer oxygen into the mixed
liquor and to provide mixing energy to keep the mixed liquor suspended solids (MLSS) in
suspension. Typically, a single device is applied both to transfer oxygen and to keep the MLSS in
suspension. For example, diffused air (both coarse and fine bubble), floating or fixed mechanical
surface aerators, and submerged turbine aerators are typical devices that could be usually applied
for the purpose of aeration.
Another important part is the clarifier, which provides two functions. One is to remove the MLSS
to produce a clarified effluent. The other is to concentrate the settled solids for return to the
bioreactor.
2.1.3 Factors affecting performance
(1) Floc-formation and filamentous growth
Since the activated sludge is composed of many types of bacteria, protozoa and small organisms,
successful operation of the systems requires development of a flocculent biomass that settles
rapidly in the clarifier, producing a dense sludge for recycle and a clear, high-quality supernatant
for discharge as treated effluent. In order to make the perfect performance, the proper proportion
of floc-forming and filamentous bacteria should be evaluated.

(2) Solids retention time (SRT)
SRT is a primary factor determining the performance of activated sludge systems. Once the SRT
was long enough for effective bioflocculation to occur, further increases had only minor effects on
soluble substrate removal. Longer SRT values may be required for the treatment of industrial
wastewaters containing more difficult to degrade materials, and may possibly be inhibitory to
biological growth. In addition, it is often designed to operate at a long SRT to achieve stabilization
of entrained organic matter and biomass or to biodegrade some slowly biodegradable organic
compounds. This can lead to limited growth of filamentous bacteria resulting in pin-point floc.
Since nitrifying bacteria are usually the most slowly growing bacteria in the system and thus the
desired SRT is determined by the minimum SRT of the most slowly growing microorganisms by
a sufficient degree to have stable performance.
(3) Mixed liquor suspended solids concentration
The performance of the activated sludge system is controlled by the mass of MLSS present.
Furthermore, the SRT for the operation is related to the mass of biomass in the system, which is
fixed once the SRT is selected. A minimum MLSS concentration is necessary to allow the
development of a flocculent biomass.
(4) Dissolved oxygen (DO)
The effect of the DO concentration in the activated sludge system on treatment performance is on
the growth of filamentous bacteria. The required DO concentration depends on the process loading
factor and specific oxygen uptake rate (SOUR). Additionally, the abilities of oxygen transfer and
mixing should also be taken into consideration to determine the performance.

(5) Nutrients
It is well known that nutrients are needed to allow the growth of biomass in biochemical operations.
However, low nutrient concentrations can favor the growth of filamentous bacteria over floc-
forming bacteria, which leads to a poor sedimentation. More severely, the less nutrients provided,
the more unbalanced growth of all bacteria.
(6) Temperature
Temperature has the main effect on the rates of biological reactions. Two additional factors must
always be considered, that is, the maximum acceptable operating temperature and the factors that
affect heat loss and gain by the process.
2.2 Sludge bulking
2.2.1 Definition
Sludge bulking occurs when the sludge fails to separate out in the sedimentation tanks (Lee and
Lin 2007). It consists of two types of sludge bulking, which are filamentous and non-filamentous
sludge bulking. It is widely accepted that the excessive growth of filamentous bacteria is the main
cause of the problem (Krhutková et al. 2002). Therefore, it refers only to filamentous sludge
bulking problem in this study.
2.2.2 Current theories to explain bulking sludge
(1) Storage selection theory

Based on recent studies, they showed that bulking sludge could have similar or even higher storage
capacity than well settling sludge (Martins et al. 2003a, b). The stored material can be metabolized
for energy generation or protein production. It would represent a strong selective advantage for
these microorganisms in competition with other filamentous and non-filamentous bacteria. A lower
storage capacity by filamentous bacteria cannot be regarded as an absolute rule in the selection
mechanism for filamentous bacteria (Martins et al. 2004).
(2) Nitric oxide (NO) hypothesis
Researchers proposed a new hypothesis for the generation of filamentous bacteria in biological
nutrients removal (BNR) systems. It is hypothesized that filamentous and floc-forming bacteria,
which are assumed to compete for organic substrate, include different intermediates of
denitrification. Nitrite and nitric oxide accumulate in the floc-forming bacteria and not in the
filamentous bacteria. Filamentous bacteria will not perform denitrification until not accumulating
the intermediate inhibiting nitric oxide. Based on this conditions, filamentous bacteria have
competitive advantages over floc-forming bacteria because they can easily utilize the slowly
biodegradable COD under aerobic conditions. The floc-forming bacteria is inhibited under aerobic
conditions with the presence of nitrite and low rate of readily biodegradable COD.
(3) Diffusion-based selection
The competition between filamentous and non-filamentous bacteria was based on the fact that the
surface-to-volume (S/V) ratio is higher for filamentous bacteria (Pipes 1967). This could give
benefits to the organisms at low substrate concentration since the mass transfer to the cells with a
high S/V ratio is more facilitated. These organisms would be led to get a relatively higher growth

rate. According to later theories, the filaments could easily penetrate outside the flocs. When the
substrate is under low concentration, the filamentous bacteria would obtain a higher substrate
concentration than the floc formers inside the floc (Sezgin et al. 1978). In diffusion-dominated
conditions, which is under low substrate concentration, filamentous open biofilm structures arise.
On the other side, compact and smooth biofilms arise at high substrate concentrations (Martins et
al. 2004). Therefore, it could be concluded that the low substrate concentration would lead to a floc
to become more open and filamentous (Martins et al. 2003a).
2.2.3 Influence factors
The influence factors involved in the sludge bulking problem could be based on the following three
parts. They are the quality of influent, environmental conditions, and operation conditions.
(1) Influent water quality
Base on a large quantities of experiments and applications, the wastewater which shows the
following aspects to determine whether it causes sludge bulking or not. Wastewater containing
high amount of carbohydrate or soluble organic compounds has significant effect on the effluent,
which shows that it is easy to lead to non-filamentous sludge bulking problem when the wastewater
contains only several suspended solids, however, more soluble and degradable organic compounds
could definitely cause severe sludge bulking. For example, the wastewater from beer, food, and
papermaking is the main source of the problem. In addition, wastewater, which consists of H2S, is
easily generates filamentous bacteria with metabolism of sulfur. Type 021N bacteria, Thiothrix are
the most common bacteria involved in this situation. Furthermore, the wastewater with low pH
value is easier to lead to sludge bulking. When pH is relatively low, filamentous fungi could
proliferate in a large amount and thus sludge bulking occurs. According to several literatures, when

pH value is lower than 6.5, it is advantageous for the growth of filamentous fungi, however,
inhibiting the growth of zoogloea (Wang et al. 2007).
(2) Environmental conditions
Several factors can be included for determining the conditions of the sludge bulking. Several
environmental factors, including pH, temperature, and nutrients, are responsible for sludge bulking.
It is well known that the growth and metabolism of microorganisms are basically based on all the
environmental conditions. Under low pH value, some fungi could rapidly proliferate and thus lead
to filamentous sludge bulking (Hu and Strom 1991). As for temperature, different filaments have
their own best living temperature in which they can reproduce exponentially. Furthermore, if
temperature is too low, the metabolic rate of microorganism in the wastewater could decrease.
Therefore a large amount of high viscosity polysaccharide is generated and lead to this specific
sludge bulking problem. The second factor is flow rate and water quality. The changeable hydraulic
loading and low dissolved oxygen concentration could probably stimulate the significant growth
of filamentous bacteria and increase sludge volume index (SVI) value, which indicate the sludge
bulking problems.
(3) Operational conditions
Three aspects of operation can be stated as follows. The influence of loading on sludge settleability
did not consistent among the research field. Some held the statement that in continuous stirred tank
system (CSTR), SVI value will decrease when the loading increases. However, in plug flow reactor
(PFR), the conclusion is opposite. Low F/M ratio was generally reported to cause sludge bulking,
which often occurs in CSTR system or some aeration basin (Wang et al. 2007). Dissolved oxygen

concentration is another essential factor that takes responsibility for sludge bulking problem. In the
aeration basin, most aerobic bacteria cannot survive under the condition of low dissolved oxygen
concentration. What’s more, filamentous bacteria can easily obtain dissolved oxygen due to its
long hypha and big surface/volume ration. The last factor is referred to sludge retention time (SRT).
According to the literatures, there is no direct relationship between SRT and sludge settleability. It
makes effect on the sludge settleability based on the other influence factors (Palm et al. 1980).
(4) Relationship between filament types and causing conditions
Filament types can be regarded as indicators of conditions causing activated sludge bulking, based
on the following conditions, which include low oxygen concentration, low F/M, septicity, nutrient
deficiency, low pH and high grease and oil. Once identification of filaments is cleared, control
methods according to the specific types of filaments causing problem could be proposed.
Table 2.1 Filament types as indicators of conditions causing activated sludge bulking.
Causative condition Filament types
Low dissolved oxygen S. natans, type 1701 and H.hydrossis.
Low organic loading>
low F/M
M. parvicella, Nocardia spp., and type 0041, 0675, 1851 and 0803.
Septic wastes/ sulfides
Thiothrix I and II, Beggiatoa spp., N. limicola II, and types 021N,
0092, 0914, 0581, 0961 and 0411.
Nutrient deficiency – N
and/or P
Thiothrix I and II, and types 021N. N. limicola III.
Low pH (<6.0) Fungi.
High grease/Oil Nocardia spp., M. parvicella and type 1863.

2.2.4 Morphological relationship between filaments and flocs
Floc-forming and filamentous bacteria exist together in the system. The relative proportion of floc-
forming and filamentous bacteria in floc determines the macrostructure (Figure 2.1). In an ideal
activated sludge floc, based on Figure 2.1 (A), the filaments provide a strong backbone around
which the well flocculated bacteria grow, which leads to a large dense floc that can settle rapidly
in the clarifier. And a clear supernatant is at the same time generated due to few small, slowly
settleable particles contained in the mixed liquor. The SVI of this type of the activated sludge is
very low. According to Figure 2.1 (B), pin-point floc consists primary individual floc particles with
little or no filamentous bacteria present to provide floc strength. This comes out the turbid
supernatant because the small and weak flocs possibly wash out from the system to the effluent.
As for the last illustration, filamentous organisms predominant the whole active sludge. This is
what we called a filamentous bulking sludge. The sludge bulking causes the filaments to extend
beyond the activated sludge flocs. They interfere with each other and make effect on settling. Thus,
the strong flocs are produced because floc enwind together by filaments. However, the floc
particles settle slowly and compact poorly, which cause the low-quality effluent (Grady Jr et al.
2011).
2.2.5 Filamentous sludge bulking control
In order to readily improve the settleability of the activated sludge system caused by excessive
filamentous bacteria, acute solutions, consisting of adding substances directly to the sludge, could
be widely used in the practice (Wanner 1994). On the other hand, since individual types of
filamentous bacteria have high affinities for different limiting nutrients, the key to controlling the
growth of filamentous organisms is to control the concentration of the growth limiting nutrient.

Figure 2.1Effect of filamentous growth on activated sludge structure: (A) ideal, non-bulking activated sludge floc; (B)
pinpoint floc; (C) filamentous bulking activated sludge (Jenkins et al. 2004).
Some filamentous bacteria have a high affinity for dissolved oxygen, some have a high affinity for
readily biodegradable organic matter, and others have a high affinity for nitrogen and phosphorus.
Therefore, they are allowed to overcome floc-forming bacteria (Grady Jr et al. 2011). There are
four groups of proposed filamentous organism. For each of them, a specific method is applied to
control the related filamentous organisms. However, it is much more economical to use nonspecific
substances such as chlorine and hydrogen peroxide to control filaments growth (Caravelli et al.
2004). Adding metal salts as coagulant is alternative nonspecific method to control the problem
(Agridiotis et al. 2007). Three factors are significant in the use of chemical oxidation to control

activated sludge bulking problem. The first is proper control of the oxidant dose. The second is
selection of an appropriate dose point. And the third is mixing at the dose point (Grady Jr et al.
2011). The adding substances are diverse and have different modes of action. Three main groups
can be distinguished: the biocides (mostly chlorine based), the ballasting agents (mostly the talc
based), the coagulating and flocculating agents (mostly synthetic polymers). The use of biocides
is to selectively kill the filamentous bacteria and then yield immediate sedimentation improvement.
Flocculating or coagulating agents are used to overcome the bridging or diffuse floc structure
associated with excessive filamentous organism growth (Jenkins et al. 2004). However, the
addition to the sludge results in the formation of larger and firmer flocs and yields immediate
sedimentation improvement. The use of ballasting agents aims at weighting the sludge, and further
reinforcing the flocs (Clauss et al. 1999).
The application of chlorine to activated sludge can be used to control the growth of filamentous
bacteria. Chlorine can oxidize filamentous bacteria faster than floc-forming bacteria, thus reduce
the quantity of the filaments in the activated sludge and influence its settling properties (Jenkins et
al. 2004). The purpose of oxidant addition is to destroy part of the activated sludge. Furthermore,
low cost and ready availability for the use of chlorine lead to a widespread application. Typical
addition range is from 2 g Cl2/(kg MLVSS·day) to a high of about 10 (Grady Jr et al. 2011).
Filamentous sludge may be destroyed by chlorine. With larger doses of chlorine the effects are
more pronounced. When chlorination is stopped the sludge will gradually tend to bulk again. Since
the results behave like this, the sludge can only be kept in a good condition by continuous dosing
with chlorine. However, during the chlorination period the effluent becomes turbid which leads to
a not desired effluent (Rensink 1974).

2.3 NZVI in wastewater treatment
2.3.1 Applications
The iron nanoparticle technology has received considerable attention for its potential application
in groundwater treatment and site remediation (Fu et al. 2014). On the other hand, the encouraging
treatment efficiencies have also been documented. Recent studies demonstrated that zero valent
iron is effective at stabilization or destruction of a host pollutants by its highly reducing character.
Several studies have demonstrated the effect of zero valent iron nanoparticles for the
transformation of halogenated organic contaminants and heavy metals. A great deal of research has
been focused on the removal of contaminants by zero-valent iron because it is non-toxic, abundant,
cheap, easy to produce, and its reduction process requires little maintenance. As for NZVI, its
higher surface are and higher reactivity than ZVI make the adaptation of NZVI to remove
contaminants more attention. In summary, NZVI is currently widely applied in the remediation and
wastewater treatment. NZVI can be utilized during the groundwater remediation and wastewater
treatment for the removal of chlorinated organic compounds, nitroaromatic compounds, arsenic,
heavy metals, nitrate, dyes, and phenol (Fu et al. 2014).
2.3.2 NZVI synthesis
Over the last several years, various synthetic methods have been developed to fabricate iron
nanoparticles. The most widely used method for environmental purposes is the borohydrate
reduction of Fe (II) or Fe (III) ions in aqueous media. The synthesis of NZVI was performed under
inert gas conditions to keep iron in its zero valent form. However, the synthesized zero valent iron
is unstable in atmospheric conditions and readily oxidized to high valent form, such as in the form
of Fe3O4, Fe2O3 (Noubactep et al. 2005). NZVI stock suspensions can freshly prepared by reducing

ferrous chloride with sodium borohydride. Deionized water and 0.2% (w/w) sodium
carboxymethyl cellulose (CMC) solution are purged with highly purified nitrogen gas for at least
twenty minutes before further use. On the other hand, 50 mL of 0.625 M ferrous chloride is
gradually added to 200 mL of 0.2% CMC solution under nitrogen gas purging. Finally, a total of
31.25 mL of 4M sodium borohydride was added drop wise to 250 mL solution containing ferrous
chloride and CMC while the solution is vigorously stirred at 1100 rpm at room temperature. The
final concentrations of NZVI and CMC in the stock solution are 0.11 M and 0.14% (w/w),
respectively. Nitrogen gas should purge throughout the synthesis process to ensure that only nano
zero valent iron is formed. The size of the nanoscaled zero valent iron synthesized by the approach
stays in the range of 55 ± 11 nm (He et al. 2007).
An improved method based on the above approach is also implemented. The end products can be
stored for a long time without being oxidized. Yuvakkumar et al. (2011) proposed the investigation
that is to synthesis zero valent iron nanoparticles in open air in presence of ethanol to prevent
massive oxidation. The reaction involved in the method is as follows (Equation 1):
2𝐹𝑒𝐶𝑙3 + 6𝑁𝑎𝐵𝐻4 + 18𝐻2 𝑂 → 2𝐹𝑒0
+ 6𝑁𝑎𝐶𝑙 + 6𝐵(𝑂𝐻)3 + 21𝐻2
The iron nanoparticles can synthesis in a flask reactor in ethanol medium with three open necks as
illustrated in Figure 2.2. For the synthesis of NZVI, 0.5406 g FeCl3·6H2O was dissolved in a 4:1
ethanol/water mixture (24 mL ethanol and 6 mL deionized water) and stirred well. At the same
time, 0.1 M sodium borohydride solution was prepared. Then the borohydride solution is poured
in a burette and added drop by drop into iron chloride solution with vigorous hand stirring. After
the first drop of sodium borohydride solution, black solid particles immediately appeared and then

Figure 2.2 Schematic diagram for synthesis of iron nanoparticles (Yuvakkumar et al. 2011).
the remaining sodium borohydride is added completely to accelerate the reduction reaction.
Another 10 minutes is needed after adding the whole borohydride solution. The filter papers are
used in filtration. The solid particles are washed three times with 25 mL portions of absolute
ethanol to remove all of the water, which prevents the rapid oxidation of zero valent iron
nanoparticles. The products are finally dried in oven at 323 K overnight. A thin layer of ethanol is
provided for storage. The size of the nanoscaled zero valent iron synthesized by the approach exists
in the range of 50-100 nm.
2.3.3 Feasibility and advantages of NZVI for sludge bulking control
Nano zero-valent iron (NZVI) is one of most commonly used engineered nanoparticles due its
specific characteristics (Lee et al. 2008, Kim et al. 2011). In addition, NZVI has been found in
wastewater treatment for nutrients removal (Shin and Cha 2008, Hwang et al. 2012). The release
of Fe2+
from the dissolution of NZVI facilitate sludge flocculation and settling as flocculants
(Wilén et al. 2004). Furthermore, NZVI has antimicrobial activity against a broad range of

microorganisms (Kim et al. 2011, Auffan et al. 2008, Kim et al. 2010) for the reason of
decomposition of cell membrane due to strong reducing conditions at the surface (Kim et al. 2010).
What’s more, since we are looking for the agent which can selectively kill filamentous bacteria
while the floc-forming bacteria will not be influenced, NZVI was reported to be a highly selective
agent (Marsalek et al. 2012b) due to the high surface/volume ratios. Filamentous bacteria are more
susceptible to NZVI exposure than floc-forming bacteria, thus resulting in selective killing
filamentous bacteria.
2.4 Research Objectives
The main objective of this research was to explore the use of NZVI for sludge bulking control and
to reduce the side effect of the use of NZVI, which is likely associated with the sludge bulking
conditions and the concentration of NZVI added into the activated sludge wastewater treatment
systems.
3. MATERIALS AND METHODS
3.1 Nano zero-valent iron synthesis and characterization
Based on the dose amount and the quality of the NZVI, the first method mentioned above is chosen
due to the following reasons. The freshness of the NZVI is very important part of the whole
research, thus there is no need for storage. The frequency of dosing is not much frequent. Above
all, we choose to fabricate NZVI freshly every time of dosing. NZVI particles were synthesized by
the sodium borohydride reduction method as reported earlier (He et al. 2007). The reaction
configuration of NZVI synthesis is shown in Figure 3.1. The reaction is shown (Equation 2):

2𝐹𝑒2+
+ 𝐵𝐻4
−
+ 3𝐻2 𝑂 → 2𝐹𝑒0
+ 𝐻2 𝐵𝑂3
−
+ 4𝐻+
+ 2𝐻2
A diluted carboxymethyl cellulose (CMC, capping agent, Sigma-Aldrich, St. Louis, MO) solution
(0.2%, w/w) served as a capping agent (Lin et al. 2010). Briefly, 200 mL of the CMC solution was
sparged with nitrogen for at least 20 min before use. Then 50 mL of freshly prepared FeCl2∙4H2O
(0.625 M) was gradually added to the CMC solution under nitrogen gas protection. Finally, a total
of 31.25 mL freshly prepared NaBH4 (4 M, Sigma-Aldrich) solution was added dropwise to the
CMC solution that was magnetically stirred at 1,100 rpm at room temperature. Nitrogen sparging
was continued for another 10 min to remove hydrogen gas. The final concentrations of NZVI in
the solution were 0.11 M. The NZVI stock suspension was purged with nitrogen gas throughout
the synthesis process to ensure that only nano-Fe0 was formed (Lee et al. 2008). The NZVI
Figure 3.1 The reaction configuration of NZVI fabrication. (1- NaBH4; 2- FeCl2; 3- N2 gas)

Figure 3.2 Transmission electron microscopic images of NZVI (Yang et al. 2013).
had an average size of 55 ± 11 nm as reported in our recent study (Yang et al. 2013). And they
were characterized by transmission electron microscopy (TEM) (Figure 3.2).
3.2 CSTR (bioreactor) setup and operation
Two identical lab-scale activated sludge systems (Tanks #1 and #2) were operated in parallel by
employing continuous stirred tank reactor systems as shown in Figure 3.4. They are common ideal
reactor types. A CSTR often refers to a model used to estimate the key until operation variables
when using a continuous agitated-tank reactor to reach a specified output. All calculations
performed with CSTR assume perfect mixing. The output composition is identical to composition
of the material inside the reactor. The CSTR is often used to simplify engineering calculations and
can be easily used to describe research reactors.
Each system involved in the research had a volume of 11.54 L and the working volume is 8.27 L
consisted of aerobic chamber and sedimentation area separated by a glass baffle. The effective

volume of the aerobic and internal settling chambers were 6.7 L and 1.57 L, respectively. For each
bioreactor, a fine bubble diffuser in conjunction with the use of a magnetic stirrer provided mixing
and aeration in the aeration chamber. The synthetic influent entered into each tank by the pump
with the same flow rate, i.e. 7.6 L/d. Each tank has an exit for effluent water. Both bioreactors were
inoculated with activated sludge obtained from the aeration basin in secondary treatment located
in Columbia WWTP (Columbia, MO) and then fed with synthetic wastewater. The aeration basin
supply large amounts of air to the mixture of primary wastewater and helpful bacteria and the other
microorganisms that consume the harmful organic matter. The growth of the helpful
microorganisms is sped up by vigorous mixing of air with the concentrated microorganisms and
the wastewater. Adequate oxygen is supplied to support the biological process at a very active level.
That is to say, the activated sludge we collected from the WWTP is in an activated condition. The
synthetic wastewater mainly contained non-fat dry milk powder with a target chemical oxygen
demand (COD) concentration of 500 mg/L. It also contained the following macro- and
micronutrients per liter: 89.18 mg NH4Cl Na2HPO4 ∙7H2O, 44 mg MgSO4, 14 mg CaCl2∙2H2O, 2
mg FeCl2∙4H2O, 3 mg MnSO4, 1.2 mg (NH4)6Mo7O24∙4H2O, 0.8 mg CuSO4, and 1.8 mg
Zn(NO3)2∙6H2O (Liang et al. 2010). The synthetic wastewater was prepared nearly every 3 days
and stored at room temperature (23 ± 1 ℃) in a covered 45 L (volume) plastic storage bin. At the
early period of the operation, 90 L influent was prepared every time. In order to confirm the
freshness of the synthetic water, the volume changed from 90 L to 45 L. There were a large quantity
of sediments in the bin due to the chemical reactions between the above chemical agents.

Figure 3.3 Schematic of operation process (1- Influent; 2- Pump; 3, 4- Mixed liquor; 5- Bubble diffuser; 6- A glass
baffle; 7- Magnetic stirring apparatus; 8- Effluent).
The bioreactors were operated and monitored for nearly 95 days after setting up, and divided into
four phases. Phase I lasted about 67 days for spontaneously sludge bulking and daily monitoring
and maintenance at the hydraulic retention time (HRT) of 0.88 days and target SRT of 10 days
associated with high bulking potential. Phase II started from day 68 onwards after the first time
NZVI dosing at the same SRT (10 days). Phase III started from day 81 onwards and lasted about
10 days after the second time NZVI dosing. Phase IV started from day 90 onwards after the third
time NZVI dosing. To determine bulking conditions, the sludge volume index (SVI) was carefully
monitored by determining the sludge settling characteristics according to the standard methods
(APHA). Through SVI measurements, microscopic observations, live and dead staining method,
an instantaneous, one-time dose of NZVI in the aeration chamber at the final concentration of 37.5

mg Fe/L in the mixed liquor was applied for sludge bulking control on day 68 for Tank #2. As for
Tank #1, it played a role as the control of the whole process. After first time dosing, second-time
consecutive dosing was made on day 81 at the concentration of 37.5 mg/L for each time, which
indicated the final concentration of 75 mg/L. Finally, a third time dosing was determined to make
at the concentration of 112.5 mg/L. The NZVI concentration was selected based on the results from
the batch study (details in the following parts).
3.3 Feedstock for CSTR systems
The feedstock applied in the system is shown in detail in Table 3.1.
Table 3.1 Feedstock for CSTR systems.
Chemical agents Concentration (mg/L) Source
Non-fat dry milk powder (COD) 500 Wal-Mart
NH4Cl 89.18 Fisher Lot# 010314
Na2HPO4∙7H2O 51.89 Fisher Lot# 034044
MgSO4 44 Fisher Lot# 897559
CaCl2∙2H2O 14 Fisher Lot# 915321A
FeCl2∙4H2O 2 Fisher Lot# 761939
MnSO4 3 Fisher Lot# 923552
(NH4)6Mo7O24∙4H2O 1.2 Fisher Lot# 985002
CuSO4 0.8 Fisher Lot# 733617
Zn(NO3)2∙6H2O 1.8 Fisher Lot# 907139
Since the synthetic wastewater should be prepared nearly every three days, the concentrated
solutions of each component were prepared in advance. According to the concentration ratios
shown in Table 3.2., it save time to prepare the feedstock for each time.

Table 3.2 Concentrated feedstock and every time dosage.
Chemical agents
Concentrated
concentration* (g/L)
Volume used each time (mL)
NH4Cl 80.26 50
Na2HPO4∙7H2O 46.7 50
MgSO4 39.6 50
CaCl2∙2H2O 12.6 50
FeCl2∙4H2O 1.8 50
MnSO4 2.7 50
(NH4)6Mo7O24∙4H2O 1.08 50
CuSO4 0.72 50
Zn(NO3)2∙6H2O 1.62 50
*Notes: the concentrated ratio is 900.
3.4 Main methods involved in the research
3.4.1 Effect of NZVI dosing on nitrifying activity
To determine the change in nitrifying bacterial activity, aliquots of mixed liquor were periodically
taken from the aeration chamber to determine the specific oxygen uptake rates (SOUR) (Hu et al.
2002). SOUR measurement is associated with oxygen uptake rate and volatile suspended solids
(VSS). It is used in measuring the metabolic activity of organisms in aquatic systems.
Microorganisms use oxygen as they consume food in an aerobic aquatic system. The rate at which
they use oxygen is an indicator of the biological activity of the system and is called the oxygen
uptake rate. High oxygen uptake rates indicate high biological activity; low oxygen uptake rates
indicate low biological activity. The analysis is based on a series of dissolved oxygen (DO)
measurements taken on a sample over a period of time. Combing oxygen uptake and volatile
suspended solids data yields a value called SOUR. SOUR describe the amount of oxygen used by
the microorganisms to consume one gram of food and is reported as mg/L of oxygen used per gram

of organic material per hour. The calculation of specific oxygen uptake rates are as follows
(Equation 3, Equation 4). The food applied in the experiment are sodium acetate and ammonia
chloride, with concentrations of 14 g/ 100 mL and 21 g/ 100 mL, respectively.
Oxygen Uptake Rates =
mg O2
L · min
× 60
min
hr
Specific Oxygen Uptake Rates = Uptake Rates ×
1000
VSS
mg
L
3.4.2 Microscopic, chemical and water quality analysis
Activated sludge in the aeration chamber of each tank was periodically subjected to light
microscopic examination (Axioskop Zeiss microscope). Every time nearly one day after NZVI
dosing into Tank #2, the activated sludge samples were subjected to live/dead analysis after
fluorescent staining with the LIVE/DEAD®
BacLightTM
bacterial viability kit (Invitrogen Co.,
Carlsbad, CA), according to the work reported elsewhere (Hu et al. 2003). The same apparatus was
used for fluorescence imaging of bacterial cells.
The influent and effluent water quality parameters such as COD (HACH, Cat.2125915, Digestion
solution for COD, high range of 20-1500 mg/L; HACH, Cat.2125815, Digestion solution for COD,
high range of 20-1500 mg/L), ammonium-N, nitrite-N, nitrate-N in the tanks were measured in
duplicate following the standard methods (APHA). The biomass concentration and properties were
also measured in duplicate following the standard methods (APHA). The parameters include COD
(Münch and Pollard 1997), MLSS, SVI, zeta potential, and particle size.

3.4.3 Filamentous bacterial DNA extraction for q-PCR analysis
Bacterial DNA samples were collected from Tank #1 and #2 on the day before and after each time
dosing, that is to say, nearly six samples has collected on day 68, 69, 81, 82, 90, and 91, respectively.
Total genomic DNA was extracted from the mixed liquor taken from the aeration chamber using a
MoBio UltraCleanTM
Soil DNA Isolation Kit (MioBio Laboratories, Inc., Carlsbad, CA). An
average of 1.0 g biomass was collected in DNA extraction. The DNA was quantified by Nanodrop
ND1000 (NanoDrop Technologies, Wilmington, NC, USA) and its purity of was analyzed by
measuring the 260/280 nm absorbance ratio. The extracted DNA samples were stored at -20˚C
before use.
Due to time limited for my research, q-PCR work should be done in the future study. Preliminary
experiments were conducted to detect a broad range of filamentous bacteria (e.g., Microthrix
parvicella, Eikelboom type 021N, Gordonia spp., Thiothrix eikelboomii) by conventional
polymerase chain reaction (PCR) methods as described elsewhere (Nielsen et al. 2004). For
quantitative microbial analysis, Type 021N was selected as a representative filamentous species
through quantitative real-time PCR (q-PCR) analysis. Type 021N stands for a large group of
filamentous bacteria and their growth is strongly related to an unbalanced influent composition and
low dissolved oxygen concentrations in the aeration chamber. Following work will be done based
on the protocol for the q-PCR analysis.

3.5 Batch study
3.5.1 Objective
Find the appropriate dosage of NZVI applied to activated sludge systems through the batch study.
3.5.2 Material and methods
(1) Materials
Four 125ml-flasks, 400 mL fresh sludge from Tank #2, freshly prepared NZVI solution with the
concentration of 0.11 M as Fe, an aeration pump, a shaker and four bubble diffusers were used in
the batch study.
In order to find the appropriate dosage of NZVI applied to the activated sludge system, batch study
is hired to determine the effect of different dosage of NZVI to the sludge. Both fresh sludge and
freshly prepared NZVI solution are used in the batch study.
A total volume of 400 mL fresh sludge is taken from one of the activated sludge tanks. Before
distributed into the flask, let the sludge stand for 30 minutes or longer if need to make the sludge
concentrated. Then remove the supernatant and distribute the concentrated sludge evenly into four
flasks and add the feedstock of the system to make the total volume to 100 mL. Among the four
units, one unit is set as negative control with only sludge and feedstock while the other units are
fed with the same mixture as that of control as well as their respective concentrations of NZVI
solution, respectively. In the 24-hour batch study procedure, NZVI solution is only applied at the
beginning of the test. Targeted concentrations were obtained by adding variable volume of NZVI
stock solution.

NZVI stock solution was freshly prepared with the mention method in the previous part. The
recommended dosage of NZVI to activated sludge is 5-25 pounds NZVI / 1000 pounds MLVSS.
After running for more than four SRTs, the activated sludge system reaches a steady state with
MLSS of 1500 mg/L. Besides, the freshly made NZVI solution has a concentration of 0.11 M,
which is equivalent to 6160 mg/L as Fe. Combined with the recommended dosage, the dosage of
NZVI applied to the system is 7.5-37.5 mg/L. The targeted concentration of NZVI and respective
volume applied are showed in Table. Since the volume of NZVI added to each flask is much smaller
than the bulk volume of sludge, it is reasonable to assume that the addition of NZVI should have
no impact on the volume of the treated unit.
Table 3.3 Targeted concentration of NZVI in batch study and respective volume applied to each flask.
Targeted NZVI concentration (mg/L)
Volume of NZVI stock solution applied to
treated unit (mL)
7.5 0.122
20 0.325
37.5 0.609
(2) Experimental procedures
① The four flasks will be fixed to a shaker to make sure the sludge and substrate mixing well
and aerator will be placed into each flask to provide oxygen for the microorganisms.
② Apply respective volume of NZVI solution to each treated unit and take samples with time
arrangement as suggested in the following Table 3.4. Take 23-hour monitoring as one trial.

Table 3.4 Time arrangement for samples.
③ Take 1 mL sludge from each unit for daily based and apply live and dead staining and then
take fluorescent microscopic images. Use software package ImageJ to analyze images
quantitatively to show the effect of respect concentration of NZVI on sludge.
Besides, after taking the sludge samples, stop the shaker and let the sludge in the unit stand for 5
minutes and then measure the COD of the supernatant. Each unit is treated with duplicate COD
samples.
④ If the effect of NZVI is not satisfying during the 23-hour monitoring, a second trial will be
applied to the batch. However, before the second batch, the sludge should have the same pre-
treatment as that in the first trial, which is concentrated and the supernatant should be removed and
then add feedstock.
Time Time interval (h)
10:00 am 1
11:00 am 2
1:00 pm 4
5:00 pm 8
1:00 am (the other day) 8
9:00 am (the other day) /

3.6 Experimental implement
Based on the previous preliminary experiments, the main process was conducted for the research.
At beginning of the experiment, daily maintenance and monitoring were made to operate the
systems and figure out how the systems performed to treat the synthetic wastewater in the lab-scale.
After the first stage of normal operation, sludge bulking problem occurred spontaneously in the
systems. Therefore, it was exactly what we expected for further research. Bulking sludge with high
SVI value was the object of the study. NZVI is the novel adding toxicant to the filamentous bulking
sludge in this research. Three times additions with different dosage were conducted during the
different periods. The first addition of NZVI with the dosage of 37.5 mg/L was added to Tank #2
when Tank #1 was the control. The corresponding parameters were also measured for monitoring
the effect of NZVI on the sludge bulking problem. Later on, the second and third time additions
were implemented for long-term inhibition of the filamentous bacteria. The second time is two-day
consecutive dosing with the concentration of 37.5 mg/L for each time. As for the third time, we
chose to dose three times as the first time dosage, which was 112.5 mg/L. The follow-up research
was also evaluated for further study.
Table 3.5 Time arrangement for every time NZVI dosing
NO. # dosing Time Day # Final concentration (mg/L)
1 4/23/2014 68 37.5
2 5/6/2014 81 75
3 5/15/2014 90 112.5

4. RESULTS AND DISCUSSION
4.1 Sludge bulking and bioreactor performance
Figure 4.1 SVI values in Tank #1 (○) and Tank #2 (◇) before NZVI dosing and in Tank #1 (●) and Tank #2 (◆) after
NZVI dosing in Tank #2 and Tank #1 as control on day 68, 81, 90,respectively.
Both bioreactors were operated at the target SRT of 10 days for the whole operation process. As
expected that long SRT operation favors filamentous bacterial growth, the SRT of 10 days could
make the bioreactors perform well under a good maintenance. However, due to the other
operational conditions, such as influent water quality, dissolved oxygen concentration, the tanks
performed worse than before, which caused the early stage of sludge bulking. These results were
shown indicated from the SVI measurements and confirmed by light microscopy (Figure 4.1). In
Tank #1, the SVI value decreased from 311 mL/g to 106 mL/g, while for Tank #2, the SVI value
decreased from 308 mL/g to 99 mL/g. The SVI values shown before indicated that the sludge we
0
100
200
300
400
500
600
700
800
900
0 10 20 30 40 50 60 70 80 90 100
SVI(mL/g)
Day of operation(day)

initially obtained from Columbia WWTP has already bulked to some extent. After domestication,
the microorganisms involved in the activated sludge adapted to the new environment, i.e. the
synthetic wastewater. It is reported that an SVI of 150 mL/g is often considered to be the dividing
line between a bulking and a non-bulking sludge (Grady Jr et al. 2011). After nearly 15 days, both
two tanks started to bulk with higher SVI values. For Tank #1, SVI increased from 282 mL/g to
629 mL/g. For Tank #2, SVI increased from 99 mL/g to 481 mL/g. Though SVI values above 150
mL/g indicate sludge bulking, the different trends in SVI change suggest the uncertainty and
complex sludge bulking mechanisms involved in each bioreactor, which leads to different bulking
Figure 4.2 Biomass COD in Tank #1 (○) and Tank #2 (◇) before NZVI dosing and in Tank #1 (●) and Tank #2 (◆)
after NZVI dosing in Tank #2 and Tank #1 as control on day 68, 81, 90,respectively. Error bars represent standard
deviation of the duplicate experiments from the mean of duplicate samples.
0
500
1000
1500
2000
2500
3000
3500
0 10 20 30 40 50 60 70 80 90 100
BiomassCOD(mg/L)
Day of operation (day)

conditions even though the two tanks were identical and operated at the same HRT and SRT.
Correspondingly, the degree of loss of sludge differed between the two bioreactors during sludge
bulking. At the beginning of the research, the average biomass COD concentration in Tank #1 and
#2 were 2,430 ± 425 mg/L and 2,475 ± 497 mg/L, respectively (Figure 4.2). There was no
significant difference in the biomass concentration between the two bioreactors. At the early period
of the process, the biomass COD concentration in both Tank #1 and #2 gradually decreased to
1,582 ± 171 mg/L and 1,744 ± 218 mg/L, respectively. Since the conditions mentioned before that
the sludge obtained had already been regarded as the bulking sludge in the early stage, the biomass
concentration gradually reduced due to a significant sludge loss in the effluent associated with
Figure 4.3 Light Microscopic images for Tank #1 (left) and Tank #2 (right) on Day 39.
sludge bulking. For comparison, Tank #2 also performed the same as Tank #1. This period could
be referred as the start-up stage for the microorganisms to adapt to the new environment. The whole
systems should go into the stable stage before the further study. Along with the evidence from SVI
measurement and microscopic observation, sludge in Tank #1 was bulking resulting in significant
sludge loss already while sludge in Tank #2 was in the early stages of bulking in the day between
Day 20 and 45 (Figure 4.3).

Figure 4.4 and 4.5 demonstrate that sludge bulking affected effluent water quality. At the SRT of
10 days before dosing (Day 1~ Day 67) and influent COD concentration of 453 ± 26 mg/L, the
effluent COD concentration from Tank #1 and #2 were 36 ± 20 mg/L and 33 ± 21 mg/L,
respectively, resulting in a similar average removal efficiency of 92% (Figure 4.4). There were also
no significant differences in effluent NH4
+
-N, NO2
-
-N or NO3
-
-N concentrations between the two
CSTR systems. The effluent NH4
+
-N concentrations from Tank #1 and #2 were 0.39 ± 0.03 mg/L
0
10
20
30
40
50
0 10 20 30 40 50 60 70 80 90 100
EffluentNO3
--NConccentration(mg/L)
(a)

Figure 4.4 Effluent NO3
-
-N (a), NO2
-
-N (b) and NH4
+
-N (c) in Tank #1 (○) and Tank #2 (◇) before NZVI dosing and
in Tank #1 (●) and Tank #2 (◆) after NZVI dosing in Tank #2 and Tank #1 as control on day 68, 81, 90,respectively.
Error bars represent standard deviation of the duplicate experiments from the mean of duplicate samples.
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 10 20 30 40 50 60 70 80 90 100
EffluentNO2
--NConccentration(mg/L)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
0 10 20 30 40 50 60 70 80 90 100
EffluentNH4
+-NConccentration(mg/L)
(b)
(c)

and 0.28 ± 0.02 mg/L, respectively, with removal efficiencies of 99%, indicating almost complete
nitrification (Figure 4.4c). Correspondingly, the effluent NO2
-
-N concentrations from Tank #1 and
#2 before NZVI dosing were 0.22 ± 0.23 mg/L and 0.13 ± 0.02 mg/L, respectively, and the effluent
NO3
-
-N concentrations were 35 ± 3 mg/L and 36 ± 3 mg/L, respectively (Figure 4.4a,b). The
effluent COD concentration before dosing in Tank #1 and #2 increased in some time (Figure 4.5),
which was mainly attributed to sludge loss in the effluent due to sludge bulking. Meanwhile, the
average effluent NH4
+
-N and NO2
-
-N concentrations increased to some degree, while the effluent
Figure 4.5 Effluent COD in Tank #1 (○) and Tank #2 (◇) before NZVI dosing and in Tank #1 (●) and Tank #2 (◆)
after NZVI dosing in Tank #2 and Tank #1 as control on day 68, 81, 90,respectively. Error bars represent standard
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80 90 100
EfflluentCOD(mg/L)

NO3
-
-N decreased. The higher effluent NH4
+
-N and NO2
-
-N concentrations were linked to its more
significant sludge bulking, suggesting that nitrifying bacteria are susceptible to perturbation
associated with filamentous sludge bulking. The water quality of both tanks were not good as
before with many obvious solids involved in the effluent.
4.2 Bioreactor performance and benefits associated with NZVI dosing
In Tank #2 with sludge bulking and sludge loss already, the use of NZVI caused different results
among the three times dosing. Based on the batch study results, we finally determined to choose
Figure 4.6 SVI vales from Tank #2 for the first time one-time dosing with NZVI dosing concentration of 37.5
mg/L.(Sample 0: before dosing, Sample 1: 2 h after dosing, Sample 2: 6 h after dosing, Sample 3: 10 h after dosing,
Sample 4: 20 h after dosing, Sample 5: 24 h after dosing).
436
429
436
429
450 452
400
420
440
460
480
500
Sample 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
SVI(mL/g)
Sample

Figure 4.7 Live and dead fluorescent images before (left) and after 24 h (right) first-time dosing of NZVI from Tank
#2. Under florescence microscopy, living cells were stained green and dead cells were stained red. After merging, the
overlap part was yellow which contained both red and green.
the dosing concentration of 37.5 mg/L. For the first time of addition, the change of the related
parameters was not much. The SVI value was still as high as 429 mL/g and even increased on the
next day, which was 450 mL/g (Figure 4.6). As for effluent water quality, effluent NO3
-
-N
concentration was the same as that of the previous day, while effluent NH4
+
-N and NO2
-
-N changed
a little after one-time dosing. Live and dead staining analysis on daily basis was also applied for
determining the visual results for the effect of NZVI dosing. As shown in the following images
(Figure 4.7), there was not much significant difference before and after dosing. Hence, the effluent
water quality and overall activated sludge bioreactor performance were only affected for a few
days. The reasons could be described as follows. (1) the concentration of NZVI was too low to
make difference; (2) the form existed in the system transferred from nano zero-valent iron to
oxidized iron, which had less reducing capacity; (3) the contact time was not long enough before
washing out.

Figure 4.8 Light microscopic images before (left) and after 24 h (right) second-time dosing from Tank #2.
Figure 4.9 SVI vales from Tank #2 for the second-time NZVI dosing with concentration of 75 mg/L.(Sample 0: before
dosing, Sample 1: 4 h after dosing, Sample 2: 16 h after dosing, Sample 3: 22 h after dosing, Sample 4 another dosing:
4 h after second-consecutive dosing, Sample 5: 16 h after second-consecutive dosing, Sample 6: 24 after second-
consecutive dosing, Sample 7: 48 h after second-consecutive dosing ).
574
600
690
643
659 667 667
789
500
550
600
650
700
750
800
850
Sample 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7
SVI(mL/g)
Sample

Therefore, another dosing plan was determined. For the second-time, the dosage was 75 mg/L of
freshly prepared NZVI and monitored for two days. However, there came the same results. No
significant change for the sludge bulking problem (Figure 4.8, 4.9).
Figure 4.10 Light microscopic images before (left) and after 24 h (right) third-time dosing from Tank #2.
Based on the previous two times addition, further dosing should be done with a larger amount of
addition. Thus, the third time dosing with the concentration of 112.5 mg/L was implemented for
Tank #2. In Tank #2 with sludge bulking and sludge loss already, the use of NZVI caused a
significant increase in effluent COD, NH4
+
-N and NO2
-
-N concentrations (Figure 4.4b, c, 4.5, 4.10).
As shown in Figure 4.10, there was almost no filaments around the flocs, thus the filaments was
selectively killed by NZVI.
Although they are short-term in nature, additional benefits of the use of NZVI included improved
sludge settling and health problem of water quality. Due to the dissolution of NZVI, the oxidized
forms (Fe2+
, Fe3+
) of iron could improve the sludge flocculation and settleability (Oikonomidis et
al. 2010), as was also confirmed in this study where the SVI was decreased after third-time NZVI
dosing.

In chlorination-based bulking control, filamentous and floc-foaming bacteria do not appear to
significantly differ in their chlorine susceptibility. Unlike chlorine, NZVI may serve as a new
bulking control agent that can selectively kill filamentous organisms, if the particle size and dose
of NZVI is adjusted such that its concentration is lethal to filaments but is much less toxic to floc-
forming bacteria. Because the unique fate and transport characteristics associated with NZVI
dissolution or agglomeration as NZVI penetrates into the floc. Thus, further research is needed to
design and test such nanomaterials for better sludge bulking control.
4.3 Impact of sludge bulking and NZVI dosing on nitrifying bacterial activity
Figure 4.11 Autotrophic SOUR values in Tank #2 before (○) and after dosing (●).Error bars represent standard
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
0 10 20 30 40 50 60 70 80 90 100
SOUR(mgO2/(gbiomass·hr))

Consistent with the effluent water quality, the autotrophic SOUR values in Tank #2 were decreased
by 40  4% due to sludge bulking on day 36. After first-time and second-time NZVI dosing, the
nitrifying bacteria activity was not affected. However, after the third-time NZVI dosing, the
nitrifying bacteria activity decreased further.
5. CONCLUSIONS
In this study, an alternative toxicant was successfully used to kill the filamentous bacteria involved
in the Columbia WWTP. Although there was no response for first two times trials, we finally found
that the positive effect of NZVI on sludge bulking control with the final dosing concentration of
nearly 112.5 mg/L. The study implied the side effect of the NZVI dosing, which includes
nitrification and effluent water quality. Therefore, the effectiveness of the biocides on controlling
the growth of filamentous bacteria should be verified by SVI monitoring, regular analysis of
effluent quality, light and fluorescent microscopic observation prior to full-scale application.
6. FUTURE STUDY
6.1 Change of some conditions before dosing
We can change some of the operational conditions for better performance. Possibly the dissolved
oxygen concentration was still enough that the NZVI could be probably oxidized and transferred
to the iron oxides, which would have weak reducing capacity or even inactivate to selectively kill
the filamentous bacteria. Since the reason like that, we can close the aeration bubble diffusers only
before NZVI dosing and make it the reducing environment.

6.2 Bioreactor performance recovery
An even longer time should be taken to evaluate the recovery time of the systems after NZVI dosing
by measuring the related water quality and sludge properties parameters, including effluent COD,
NH4
+
-N, NO2
-
-N, NO3
-
N, sludge COD, sludge MLSS, SOUR, and SVI. Based on the results, the
effect of NZVI will be studied, which indicates whether the addition of NZVI to the bulking sludge
is a long-term or an instantaneous approach.
6.3 Sludge bulking associated with long changeable SRT operation
Since the reactors are operated for a long time, two phases of operation period could be made for
the purpose of finding out the effect of different SRT on the sludge bulking. The research plan
could be described as follows. Both reactors are initially operated at the target SRT of 10 days for
about two months, then followed by the operation that the SRT is increased to 20 days. As expected
that long SRT operation favors filamentous bacterial growth (Grady Jr et al. 2011), an increase in
SRT from 10 to 20 days encouraged the growth of filamentous bacteria as indicated from the SVI
measurements and could also be further confirmed by light microscopy.
6.4 Impact of sludge bulking and NZVI dosing on nitrifying bacterial population
The side effect of NZVI dosing in activated sludge is inferred from its effect on the growth of
sensitive nitrifying bacteria, which include ammonia-oxidizing bacteria (AOB) and nitrite-
oxidizing bacteria (NOB). In order to analyze the impact of NZVI on nitrifying population, the
collected bacterial DNA samples should be analyzed by Terminal Restriction Fragment Length
Polymorphism (T-RFLP) targeting the 16S RNA genes of AOB (Mobarry et al. 1996) and NOB

(Regan et al. 2002). Based on the results, we could also find the change in nitrifying bacterial
community structure in the tank before and after NZVI dosing. The dominant bacteria among AOB
and NOB will be known according to the analysis. We could figure out how NZVI make effect on
the nitrifying bacterial population.
6.5 Identification of filamentous bacteria before and after NZVI dosing
Since filamentous bacterial DNA samples were collected and stored at -20˚C, further q-PCR
analysis should be done for identification of specific filamentous bacteria before and after NZVI
dosing. Preliminary experiments can be conducted to detect a broad range of filamentous bacteria
by conventional polymerase chain reaction methods. In our research group, a recent result showed
that there commonly existed Type 021N in the sludge bulking systems which were operated in the
same conditions. For quantitative microbial analysis, Type 021N could be selected as a
representative filamentous species through quantitative real-time (q-PCR) analysis. Type 021N
stands for a large group of filamentous bacteria and their growth is related to an unbalanced influent
composition and low oxygen concentrations in aeration tanks. The q-PCR assays are performed
with the system, according to the protocols. Based on the results, identification of filamentous
bacteria could be completed and quantitative analysis can enhance the research conclusions.

ACKNOWLEDGEMENTS
My deepest gratitude goes first and foremost to my advisor and mentor Dr. Zhiqiang Hu for the
continuous support of my studies during the senior year, for his patience, motivation, enthusiasm,
encouragement and immense knowledge. His guidance helped me in all the time of research.
Without his illuminating instruction and persistent help this thesis would not have been possible.
Besides, I am grateful to my fellow lab mates: Can Cui for her persistent hard-working with me.
Shengnan Xu for her generous advice on my research. Tianyu Tang for helping me with some basic
lab work. Minghao Sun for helping me learn how to do all inorganic measurement of water quality
and SOUR. Jianyuan Xu and Chiqian Zhang for developing the protocol of DNA extraction. Thanks
to Meng Xu, Jialiang Guo, Wenna Hu, Jingjing Dai and Meng Xu, for all the help and great time
we have had in the last one year.
Last but not least, thanks to my beloved family and my dear friends for their loving considerations,
support and encouragement throughout this entire process. I am so blessed to have you by my side.

DEDICATION
I dedicate this thesis to my beloved parents, whose moral encouragement and support helped me
realize my bachelor’s degree goal.

7. REFERENCES
Eikelboom, D.H., Andreadakis, A. and Andreasen, K. (1998) Survey of filamentous populations
in nutrient removal plants in four European countries. Water Science and Technology 37(4-5), 281-
289.
Martins, A.M.P., Pagilla, K., Heijnen, J.J. and Van Loosdrecht, M.C.M. (2004) Filamentous
bulking sludge - A critical review. Water Research 38(4), 793-817.
Clauss, F., Balavoine, C., Hélaine, D. and Martin, G. (1999) Controlling the settling of activated
sludge in pulp and paper wastewater treatment plants, pp. 223-229.
Krhutková, O., Ruzicková, I. and Wanner, J. (2002) Microbial evaluation of activated sludge and
filamentous population at eight Czech nutrient removal activated sludge plants during year 2000,
pp. 471-478.
Kappeler, J. and Gujer, W. (1994) Influences of wastewater composition and operating conditions
on activated sludge bulking and scum formation. Water Science and Technology 30(11), 181-189.
Guo, J., Peng, Y., Wang, Z., Yuan, Z., Yang, X. and Wang, S. (2012) Control filamentous bulking
caused by chlorine-resistant Type 021N bacteria through adding a biocide CTAB. Water Research
46(19), 6531-6542.
Lemmer, H. (2003) Book Review: Manual on the Causes and Control of Activated Sludge Bulking,
Foaming and other Solids Separation Problems von D. Jenkins, M. G. Richard and G. T Daigger.
Lou, I. and Zhao, Y. (2012) Sludge bulking prediction using principle component regression and
artificial neural network. Mathematical Problems in Engineering 2012.
Eikelboom, D.H. (2000) Process control of activated sludge plants by microscopic investigation,
IWA publishing.
Jenkins, D., Richard, M.G. and Daigger, G.T. (2004) Manual on the causes and control of activated
sludge bulking, foaming, and other solids separation problems, IWA publishing.
Wanner, J. (1994) Activated sludge: bulking and foaming control, CRC Press.

Guo, J., Peng, Y., Peng, C., Wang, S., Chen, Y., Huang, H. and Sun, Z. (2010) Energy saving
achieved by limited filamentous bulking sludge under low dissolved oxygen. Bioresource
Technology 101(4), 1120-1126.
Bitton, G. (2010) Wastewater Microbiology: Fourth Edition.
Ramírez, G.W., Alonso, J.L., Villanueva, A., Guardino, R., Basiero, J.A., Bernecer, I. and
Morenilla, J.J. (2000) A rapid, direct method for assessing chlorine effect on filamentous bacteria
in activated sludge. Water Research 34(15), 3894-3898.
Wimmer, R.F. and Love, N.G. (2004) Activated sludge deflocculation in response to chlorine
addition: The potassium connection. Water Environment Research 76(3), 213-219.
Séka, M.A., Kalogo, Y., Hammes, F., Kielemoes, J. and Verstraete, W. (2001) Chlorine-
Susceptible and Chlorine-Resistant Type 021N Bacteria Occurring in Bulking Activated Sludges.
Applied and Environmental Microbiology 67(3-12), 5303-5307.
Maynard, A.D., Aitken, R.J., Butz, T., Colvin, V., Donaldson, K., Oberdörster, G., Philbert, M.A.,
Ryan, J., Seaton, A., Stone, V., Tinkle, S.S., Tran, L., Walker, N.J. and Warheit, D.B. (2006) Safe
handling of nanotechnology. Nature 444(7117), 267-269.
Jortner, J. and Rao, C.N.R. (2002) Nanostructured advanced materials. Perspectives and directions.
Pure and Applied Chemistry 74(9), 1491-1506.
Crane, R.A. and Scott, T.B. (2012) Nanoscale zero-valent iron: Future prospects for an emerging
water treatment technology. Journal of Hazardous Materials 211-212, 112-125.
Elliott, D.W., Lien, H.L. and Zhang, W.X. (2009) Degradation of lindane by zero-valent iron
nanoparticles. Journal of Environmental Engineering 135(5), 317-324.
Lee, C., Jee, Y.K., Won, I.L., Nelson, K.L., Yoon, J. and Sedlak, D.L. (2008) Bactericidal effect
of zero-valent iron nanoparticles on Escherichia coli. Environmental Science and Technology
42(13), 4927-4933.
Yuvakkumar, R., Elango, V., Rajendran, V. and Kannan, N. (2011) Preparation and
characterization of zero valent Iron nanoparticles. Digest Journal of Nanomaterials and
Biostructures 6(4), 1771-1776.

Shin, K.H. and Cha, D.K. (2008) Microbial reduction of nitrate in the presence of nanoscale zero-
valent iron. Chemosphere 72(2), 257-262.
Chang, N.B., Wanielista, M., Hossain, F., Zhai, L. and Lin, K.S. (2008) Integrating nanoscale zero-
valent iron and titanium dioxide for nutrient removal in stormwater systems. Nano 3(4), 297-300.
Wilén, B.M., Keiding, K. and Nielsen, P.H. (2004) Flocculation of activated sludge flocs by
stimulation of the aerobic biological activity. Water Research 38(18), 3909-3919.
Marsalek, B., Jancula, D., Marsalkova, E., Mashlan, M., Safarova, K., Tucek, J. and Zboril, R.
(2012a) Multimodal action and selective toxicity of zerovalent iron nanoparticles against
cyanobacteria. Environmental Science and Technology 46(4), 2316-2323.
Ardern, E. and Lockett, W.T. (1914) Experiments on the oxidation of sewage without the aid of
filters. Journal of the Society of Chemical Industry 33(10), 523-539.
Grady Jr, C.L., Daigger, G.T., Love, N.G., Filipe, C.D. and Leslie Grady, C. (2011) Biological
wastewater treatment, IWA Publishing.
Lee, C. and Lin, S. (2007) Handbook of Environmental Engineering Calculations 2nd Ed,
McGraw-Hill Prof Med/Tech.
Martins, A.M.P., Heijnen, J.J. and Van Loosdrecht, M.C.M. (2003a) Effect of feeding pattern and
storage on the sludge settleability under aerobic conditions. Water Research 37(11), 2555-2570.
Martins, A.M.P., Heijnen, J.J. and Van Loosdrecht, M.C.M. (2003b) Effect of dissolved oxygen
concentration on sludge settleability. Applied Microbiology and Biotechnology 62(5-6), 586-593.
Pipes, W.O. (1967) Bulking of activated sludge. Adv Appl Microbiol 9, 185-234.
Sezgin, M., Jenkins, D. and Parker, D.S. (1978) A unified theory of filamentous activated sludge
bulking. Journal of the Water Pollution Control Federation 50(2), 362-381.
Wang, F.X., Long, T.R. and Guo, J.S. (2007) Study on factors affecting the activated sludge
bulking and its control. Chongqing Jianzhu Daxue Xuebao/Journal of Chongqing Jianzhu
University 29(1), 117-121.

Hu, P. and Strom, P.F. (1991) Effect of pH on fungal growth and bulking in laboratory-activated
sludges. Research Journal of the Water Pollution Control Federation 63(3), 276-277.
Palm, J.C., Jenkins, D. and Parker, D.S. (1980) Relationship between organic loading, dissolved
oxygen concentration and sludge settleability in the completely-mixed activated sludge process.
Journal of the Water Pollution Control Federation 52(10), 2484-2506.
Caravelli, A., Giannuzzi, L. and Zaritzky, N. (2004) Effect of chlorine on filamentous
microorganisms present in activated sludge as evaluated by respirometry and INT-dehydrogenase
activity. Water Research 38(9), 2394-2404.
Agridiotis, V., Forster, C.F. and Carliell-Marquet, C. (2007) Addition of Al and Fe salts during
treatment of paper mill effluents to improve activated sludge settlement characteristics.
Bioresource Technology 98(15), 2926-2934.
Rensink, J.H. (1974) New approach to preventing bulking sludge. Journal of the Water Pollution
Control Federation 46(8), 1888-1894.
Fu, F., Dionysiou, D.D. and Liu, H. (2014) The use of zero-valent iron for groundwater remediation
and wastewater treatment: A review. Journal of Hazardous Materials 267, 194-205.
Noubactep, C., Meinrath, G., Dietrich, P., Sauter, M. and Merkel, B.J. (2005) Testing the suitability
of zerovalent iron materials for reactive walls. Environmental Chemistry 2(1), 71-76.
He, F., Zhao, D., Liu, J. and Roberts, C.B. (2007) Stabilization of Fe-Pd nanoparticles with sodium
carboxymethyl cellulose for enhanced transport and dechlorination of trichloroethylene in soil and
groundwater. Industrial & Engineering Chemistry Research 46(1), 29-34.
Kim, J.Y., Lee, C., Love, D.C., Sedlak, D.L., Yoon, J. and Nelson, K.L. (2011) Inactivation of
MS2 coliphage by ferrous ion and zero-valent iron nanoparticles. Environmental Science and
Technology 45(16), 6978-6984.
Hwang, Y.H., Ahn, Y.T. and Shin, H.S. (2012) Recovery of ammonium salt from nitrate-
containing water by iron nanoparticles and membrane contactor. Environmental Engineering
Research (EER) 17(2), 111-116.
Auffan, M., Achouak, W., Rose, J., Roncato, M.-A., Chanéac, C., Waite, D.T., Masion, A., Woicik,
J.C., Wiesner, M.R. and Bottero, J.-Y. (2008) Relation between the redox state of iron-based

nanoparticles and their cytotoxicity toward Escherichia coli. Environmental Science & Technology
42(17), 6730-6735.
Kim, J.Y., Park, H.-J., Lee, C., Nelson, K.L., Sedlak, D.L. and Yoon, J. (2010) Inactivation of
Escherichia coli by nanoparticulate zerovalent iron and ferrous ion. Applied and Environmental
Microbiology 76(22), 7668-7670.
Marsalek, B., Jancula, D., Marsalkova, E., Mashlan, M., Safarova, K., Tucek, J. and Zboril, R.
(2012b) Multimodal action and selective toxicity of zerovalent iron nanoparticles against
cyanobacteria. Environmental Science & Technology 46(4), 2316-2323.
Lin, Y.-H., Tseng, H.-H., Wey, M.-Y. and Lin, M.-D. (2010) Characteristics of two types of
stabilized nano zero-valent iron and transport in porous media. Science of the Total Environment
408(10), 2260-2267.
Yang, Y., Guo, J. and Hu, Z. (2013) Impact of nano zero valent iron (NZVI) on methanogenic
activity and population dynamics in anaerobic digestion. Water Research 47(17), 6790-6800.
Liang, Z., Das, A. and Hu, Z. (2010) Bacterial response to a shock load of nanosilver in an activated
sludge treatment system. Water Research 44(18), 5432-5438.
APHA, A. AEF (1998) Standard methods for the examination of water and waste water,
Washington, DC.
Hu, Z., Chandran, K., Grasso, D. and Smets, B.F. (2002) Effect of nickel and cadmium speciation
on nitrification inhibition. Environmental Science and Technology 36(14), 3074-3078.
Hu, Z., Chandran, K., Grasso, D. and Smets, B.F. (2003) Impact of metal sorption and
internalization on nitrification inhibition. Environmental Science & Technology 37(4), 728-734.
Münch, E.v. and Pollard, P.C. (1997) Measuring bacterial biomass-COD in wastewater containing
particulate matter. Water Research 31(10), 2550-2556.
Nielsen, P., Thomsen, T. and Nielsen, J. (2004) Bacterial composition of activated sludge-
importance for floc and sludge properties. Water Science & Technology 49(10), 51-58.

Oikonomidis, I., Burrows, L.J. and Carliell-Marquet, C.M. (2010) Mode of action of ferric and
ferrous iron salts in activated sludge. Journal of Chemical Technology and Biotechnology 85(8),
1067-1076.
Mobarry, B.K., Wagner, M., Urbain, V., Rittmann, B.E. and Stahl, D.A. (1996) Phylogenetic
probes for analyzing abundance and spatial organization of nitrifying bacteria. Applied and
Environmental Microbiology 62(6), 2156-2162.
Regan, J.M., Harrington, G.W. and Noguera, D.R. (2002) Ammonia-and nitrite-oxidizing bacterial
communities in a pilot-scale chloraminated drinking water distribution system. Applied and
Environmental Microbiology 68(1), 73-81.

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