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 water Raw 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 + 18𝐻2𝑂 → 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− + 3𝐻2𝑂 → 2𝐹𝑒0 + 𝐻2𝐵𝑂3

− + 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

SV

I(m

L/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

Bio

mas

s C

OD

(m

g/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

Eff

luen

t N

O3

- -N

C

oncc

entr

atio

n(m

g/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

Eff

luen

t N

O2- -

N

Co

ncc

entr

atio

n (

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

Eff

luen

t N

H4+-N

C

oncc

entr

atio

n (

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

Eff

lluen

t C

OD

(m

g/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

450452

400

420

440

460

480

500

Sample 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

SV

I (m

L/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

643659 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

SV

I (m

L/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

SO

UR

(m

g O

2/(

g b

iom

ass·

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.


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