Fate of enteric viruses and bacteriophages in sequencing batch reactor treating domestic wastewater

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY --- NGUYEN HOANG PHUONG THAO FATE OF ENTERIC VIRUSES AND BACTERIOPHAGES IN SEQUENCING BATCH REACTOR TREATING DOMESTIC WAST

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY -

NGUYEN HOANG PHUONG THAO

FATE OF ENTERIC VIRUSES AND

BACTERIOPHAGES IN SEQUENCING BATCH REACTOR TREATING DOMESTIC

WASTEWATER

MASTER'S THESIS

Hanoi, 2018

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

- -

NGUYEN HOANG PHUONG THAO

FATE OF ENTERIC VIRUSES AND

BACTERIOPHAGES IN SEQUENCING BATCH REACTOR TREATING DOMESTIC

WASTEWATER

MAJOR: ENVIRONMENTAL ENGINEERING

SUPERVISORS:

Assoc Prof HIROYUKI KATAYAMA

Prof HIROAKI FURUMAI

Dr PHAM VAN QUAN Assoc Prof NGUYEN MANH KHAI

Hanoi, 2018

i

ACKNOWLEDGEMENT

Master thesis is completed at the Vietnam Japan University A completed study would not be done without any assistance Therefore, the author who conducted this thesis gratefully gives acknowledgment to their support and motivation during the time of doing this research

I would first like to express my endless thanks and gratefulness to my supervisor Associate Professor Hiroyuki Katayama, Professor Hiroaki Furumai, Associate Professor Nguyen Manh Khai and Doctor Pham Van Quan His kindly support and continuous advices went through the process of completion of my thesis His encouragement and comments had significantly enriched and improved my work Without his motivation and instructions, the thesis would have been impossible to

I wish to receive the contribution, criticism of the professors

Sincerely thank

ii

TABLE OF CONTENTS

INTRODUCTION……….1

Research purposes and significance 1

Scope and objective 2

CHAPTER 1 LITERATURE REVIEW……….3

1.1 Review on enteric viruses 3

1.1.1.Sources of enteric viruses in the environment 3

1.1.2.Virus transport and survival in the environment 4

1.1.3.Waterborne enteric viruses and potential human diseases 5

1.1.4.The use of microbial indicator 9

1.2 Review on bacteriophages 10

1.2.1.The value of phages as surrogates for enteric viruses 11

1.2.2.F-specific RNA bacteriophages 12

1.2.3.Detected method for virus and phages 13

1.3 Review on wastewater treatment plant 18

1.3.1 State of wastewater treatment plant in the world and in Vietnam 18

1.3.2 Sequencing batch reactor treatment 19

CHAPTER 2 MATERIALS AND METHODS……… 23

2.1 Site description 23

2.2 Sample collection 24

2.3 Several indicators of analysis 26

iii

2.4 Sample concentration 28

2.5 Detection of E.coli and total coliforms by plate count 28

2.6 Detection of F-RNA phages by plaque assay 29

2.7 Method for quantitative genotyping of infectious FRNA phages coupled with MPN method 29

2.8 Recovery of viruses from sludge method 30

2.9 RT-PCR methods 31

2.10 Statistical analysis 34

CHAPTER 3 RESULTS AND DISCUSSION……….36

3.1 The occurrence of viruses and F-RNA phages 36

3.2 Reduction of viruses, F-RNA phages and microbial indicators 39

CHAPTER 4 CONCLUSION……… 45

REFERENCE ……… 46

iv

LIST OF TABLES

Table 1.1 Advantage and disadvantage of culture and molecular methods 18

Table 2.1 The reaction mixture for RT 32

Table 2.2 The thermal condition of RT reaction 32

Table 2.3 The reaction mix for PCR 32

Table 2.4 The thermal condition for PCR 33

Table 3.1 Log10 mean concentration of F-RNA specific genotype 37

Table 3.2 Log10 concentration of E.coli and total coliforms 38

v

LIST OF FIGURES

Figure 2.1 Influent tank 23

Figure 2.2 Reaction tank 23

Figure 2.3 Decanter 24

Figure 2.4 Effluent area 24

Figure 2.5 The cycle of Sequencing Batch Reactor

Figure 2.6 Using a long bucket to take the sample 25

Figure 2.7 Using a syringe to take the sample 26

Figure 2.8 Thermal cycler 34

Figure 2.9 7500 fast Real-time PCR system 34

Figure 3.1 Concentration of E.coli 40

Figure 3.2 The concentration of total coliforms 40

Figure 3.3 The concentration of F-RNA phages genotype (a – GI, b-GII, c-GIII, d-GIV) 41

Figure 3.4 Log 10 reductions of F-RNA phages genotype GI, GII, GIII, GIV and microbial indicators 43

vi

LIST OF ABBREVIATIONS

1

INTRODUCTION

Research purposes and significance

Viruses cause harm to humans because they are very small in size and therefore move in water and can cause disease with very low dose A group of viral pathogens, from within the human body, excrete through the feces, and over-disperse the drainage pipes Currently, researchers have identified several types of enteric viruses in the domestic effluent (Wong et al., 2012) mainly including enteroviruses (EVs), rotaviruses (RVs), adenoviruses (AdVs), noroviruses (NVs) hepatitis A virus (HAV) and astroviruses (AVs) The virus is responsible for some infectious diseases such as gastroenteritis, conjunctivitis, and respiratory disease, both developed and developing countries throughout the world Therefore, studying the behavioral characteristics, existence or inactivated of the viruses in the water treatment stages of the plant is the most important

It is complicated and expensive to analyze all types of virus At present, many previous studies have tried to identify suitable viral indicators of wastewater treatment efficiency Several studies have proposed F- specific coliphages can be a good indicator monitoring the virus removal from wastewater (Tree et al., 2003; Duran et al., 2003), because there have similar morphological characteristics with many enteric viruses suggesting that they have the same single-stranded RNA, icosahedral shape, less than 50nm, and they are more persistent than bacteria F-

Specific coliphages are bacteriophages that infect Escherichia coli cells These

FRNA coliphages include MS-2 in group I, GA in group II, Qβ in group III and SP

in group IV

GII and GIII F-RNA phage genotypes was found primarily from human feces, while

GI and GIV F-RNA phage genotype are involved in animal waste (Vinjé et al., 2004) The use of F- specific coliphages as indicators of the presence and behavior

of enteric viruses and animal enteric viruses has always been attractive because of

2

the easy of detection and low cost associated with plaque assay and for similarity to enteric viruses in terms of transport and survival characteristics

Scope and objective

This research studied the fate of enteric viruses and bacteriophages during the phase

of sequencing batch reactor treatment plant Sequencing batch reactor (SBR) is a fill and draws activated sludge system In this system, each tank in the SBR system is filled during a discrete period of time and then operated as a batch reactor It means, once the reactor is full, it behaves like a conventional activated sludge system, but without a continuous influent or effluent flow The aeration and mixing are discontinued after the biological reactions are complete, the biomass settles, and the treated supernatant is removed The reason why this study chooses SBR system because the hydraulic retention time is time-based so there is no short-circuiting The influent and effluent can be pair so it is easy to observe the treatment efficiency using only a grab sample In addition, almost no research data in sequencing batch reactor for viruses’ removal So SBR system is the good target for an assessment of virus

This study has three main objectives:

1 To investigate the removal of enteric viruses and F-RNA phages in SBR system

2 To evaluate the concentration of enteric viruses and F-RNA phages in activated sludge

3 To find out the relationship between FRNA phage specific group I, II, III, IV and enteric viruses

3

CHAPTER 1 LITERATURE REVIEW

1.1 Review on enteric viruses

Viruses are infectious agents that multiply only when they are inside a living organism of host cells Viruses can infect all types of organisms, from animals, plants, bacteria, and archaea Enteric viruses are the virus that can multiply in the gastrointestinal of humans and animals

In general, viruses are smaller in size than bacteria Most of the viruses studied have

a diameter ranging from 20 to 300 nanometers The virus is a macromolecular structure, no energy-producing system, no ribosome, no individual growth, no division and no susceptibility to antibiotics, contains only one of two types of nucleic acid (DNA or RNA), living endocytic parasites

1.1.1 Sources of enteric viruses in the environment

Enteric viruses are viruses that multiply in the intestines of humans and animals and are emitted by the environment through feces and large numbers excrete from infected humans An infected person can emit more than 1 billion viruses/g of feces And from human sources, viruses will migrate to other environments through water pipelines, sewage treatment plants, or direct emissions to the environment

Usually, enteric viruses are found in surface water and the source can be from household effluents or urban waste water Several studies have shown that enteric viruses such as rotaviruses, HAVs, HEVs, enteroviruses and astroviruses are present

in surface water, groundwater and sewage effluents (Heerden et al., 2005) Sedmak

et al., 2005 has been shown that in the wastewater environment the concentration of the pathogenic viruses is about 100-10,000 infectious units/L The concentration of

1 to 10 infectious units/L in surface water In the groundwater environment, this amount is in the range of 0-200 infectious virus /100L, according to the US.EPA,

b, Survival

Although the virus can not multiply outside the host cell and cannot grow outside of the environment, it can live or exist inside the environment for a certain period of time from 28 to 88 days (Schimdt et al., 1969) The existence of the virus depends

on several factors, such as the characteristic of the virus, water characteristics such

as pH, turbidity, level of disinfection Most viruses are inactivated at 56 ° C for 30 minutes, or at 100 ° C for a few seconds The virus is also stable at cold temperatures and can be stored for up to -70C Viruses are inactivated by ultraviolet light and in environments that contain oxidants and reducing agents such as formaldehyde, chlorine, iodine, and H2O ß-Propiolactone and formaldehyde are chemicals used to inactivate viruses in vaccine production (Aimee et al., 2015)

In wastewater, there are some types of pathogenic microorganisms, especially in areas where there is no proper sanitation system Microorganisms and viruses often

do not live long in wastewater because they are not suitable environments, but they exist for a certain period of time depending on the species to infect people, animals, and plants Some types of microorganisms and viruses as follows:

- Intestinal bacteria (E.coli), can live in dirty water for 9-14 days at a

temperature of 20-220C (Elsas et al., 2011)

5

- The Adenovirus, Echo, Coxsackie virus live up to 15 days (Artur and Nigle, 2006)

1.1.3 Waterborne enteric viruses and potential human diseases

Viruses are important pathogens infecting to humans, animals, plants, and microorganisms The pathogenic virus only transmits the disease to each specific host In almost, viruses infect humans and will not infect animals and plants In contrast, viruses that infect animals will not infect humans Only a few enteric viruses are contagious for both humans and animals

Viruses infiltrate through many ways, but the main route is the digestive tract, the virus is excreted in the feces of the infected person, sometimes from the vomit More than 140 different types of enteric viruses have been studied that can infect humans Several common type of viruses are transmission of the virus such as noroviruses, hepatitis A virus (HAV), hepatitis E virus (HEV), rotaviruses and enteroviruses

a, Norovirus

Norovirus belongs to the family Caliciviridae and has a viral RNA These viruses are usually small and have no surrounding envelope, sRNA Currently, NoVs are classified into five different groups of genes: GI, GII, GIII, GIV and GV NoVs are

in the GI, GII and GIV groups, but most human pathogens are GI and GII The Norwalk virus was found under a microscope in 1978 from a patient's stool sample

in Norwalk, Ohio This virus can live very harshly in harsh environmental conditions as it can live yearly in frozen or hot temperatures of 60 ° C for 30 minutes or in chloride 6.25 mg/liter (Knight et al., 2016)

Norovirus potentially highly infectious Viruses are present in the stool and in the vomit liquid Norovirus can be spread through contacts between healthy people and infected people, such as sharing eating habits, sleeping, grasping supplies used by people who carry the disease or using the same ingested or contaminated by

6

norovirus In the United States and Canada, many Norwalk virus outbreaks have occurred on cruise ships It is also common in nursing homes, nursing homes for the elderly, in kindergartens and schools, etc Older people with weakened immune systems, pregnant women and children may be more likely to become infected than other objects (Knight et al., 2016)

As estimated by the Centers for Disease Control and Prevention 2012, at least 50%

of cases of acute gastroenteritis are attributed to norovirus The National Institutes

of Health said that between 600,000 and 1 million people in the country are infected with norovirus annually

b, Hepatitis virus

Hepatite A virus, belongs to the family Picornaviridae and belongs to type 72 of

Enterovirus and second ranks in foodborne botulism Compared with other enteroviruses, HAV is relatively stable at room temperature, at a temperature of 60°

C The virus is only partially inactivated while boiling for 5 minutes causes inactivation of the virus Hepatitis A virus can persist in water for 3 to 10 months HAV is resistant to organic solvents such as ether, acid HAV multiply in the hepatocyte, the infected cell releases the viral particles infects the bloodstream and

is then released into the stool

Transmission occurs between humans and humans by eating food and drinking contaminated water Hepatitis by HAV is widespread in densely populated areas Diseases occur all over the world which usually occurs in late autumn and early winter The disease occurs in all ages, children with the benign or asymptomatic disease, the older the infection the heavier

Hepatite E virus was found in 1955 during a pandemic in New Delhi, India

Hepatitis E is found all over the world, but most are in tropical countries near the equator These countries include Latin America, Africa, the Indian mainland, the Middle East, and Asia, especially those in Southeast Asia Periodic outbreaks, approximately 5 to 10 years, in heavy rainy seasons, resulting in flooding

7

Hepatitis E virus is not transmitting by blood, sex, and mother to child, mainly through gastrointestinal (hepatitis A) Because HEV virus occurs in the feces, garbage, wastewater, and when the floods happen the virus attached to food and drinking water When we eat the food or drink will be infected A healthy person, if exposed to the feces of an infected person, is not thoroughly hygienically cleaned, then he or she may be exposed to a virus or drink unboiled water, the uncooked meat of infected animals will also be infected Between 1986 and 1988, more than 120,000 people living in China's Xinjiang region were "poisoned" for hepatitis E Even in the United States, hepatitis E is the cause of more than 50% of acute hepatitis (Hongwei et al., 2010)

c, Rotaviruses

The complete rotaviruses are about 60-80 nm in diameter, like a short thoracic wheel Rotaviruses contain 11 double-stranded RNA, approximately 18,000 pairs of basalts

Rotaviruses can survive for several days at 400C, even at 200C in the presence of CaCl2 rotaviruses that remain infectious for months At -200C the virus can survive for many years Rotaviruses infect small intestinal villi, which alter the structure and function of the epithelium Three layers of protein capsid help the virus resist acid pH in the stomach and digestive enzymes in the intestines (Bruce et al., 1983) Diarrhea caused by rotavirus is very contagious, mainly through the fecal-oral and oral-oral route The amount of virus excreted in the feces is very sick so easily spread to other children and caregivers In addition, rotavirus can survive in the natural environment such as on contact surfaces of toys, furniture, floors, household items

Around the world, about 2 million cases have been hospitalized each year for acute diarrhea caused by rotavirus Over the five years (1973-1978), rotaviruses have been identified as the most common cause of acute diarrhea in infants and young children around the world, especially children under 5 years of age Rotavirus

Viruses are unlikely to infect when heated to 50 ° C Enterovirus can be stable in thawing temperatures for decades, at 4 ° C for weeks, and in heat room for several days Viruses in Stool Stools at room temperature for at least 4 weeks When dried, enterovirus loses its infection capacity They are also inactive when treated with ultraviolet light, dye to detect them under the microscope (Chen et al., 2008)

Viruses enter the body through the digestive tract and focal in the stool and pharynx The common feature of the viruses in this group is the reproduction and spread from the carrier to the healthy person through the digestive tract or respiratory tract Invasive oncogenes and lymph nodes in the throat When it reaches the required amount, the virus enters the bloodstream Patients will have the fever (about 1 week after the virus enters the body) Humans are the only reservoir

of Enterovirus Children are more likely to be infected with Enterovirus than adults

In the summer of 2000-2001, an outbreak of EV71 occurred in Sydney, with 200 children hospitalized, including nine patients with CNS and five pulmonary edemata EV71 has been identified as a causative agent in all patients with pulmonary edema (Nolan et al., 2003)

In 2009, EV cases in China reached 1,155,525 cases, of which 13,810 (1.2%) were serious cases and 353 (0.03%) deaths Cases are widely distributed throughout

9

China Among EV71 cases, EV71 accounts for 41% of cases, accounting for 81%

of all serious cases and accounting for 93% of deaths (WHO, 1979)

1.1.4 The use of microbial indicator

Contaminated water is mainly caused by feces excrete from human or animal In the feces there have many microorganisms are excreted from the intestines or other parts of the human body, in addition to some of the viruses mentioned above, there are many other pathogens exist in human and animal feces

Until now, coliforms bacteria have still considered an indicator for water contaminant Coliforms bacteria are organisms that are present in the environment and are present in the feces of hot-blooded animals and humans Coliforms bacteria

do not cause disease Coliforms bacteria have similar characteristics to intestinal microbes, such as resistant in the environment, morphological characteristics, and size, so their presence in drinking water indicates that pathogenic organisms may be present Testing the presence of coliform bacteria is easy and simple compared to testing with all possible pathogens

There are three different groups of coliforms bacteria, each with a different risk level: total coliform, fecal coliform, and Escherichia coli To indicate that the

environment is contaminated with feces, E.coli and fecal coliforms are used as an

indicator microorganism, which appears in large numbers in the human intestine and human feces

In the past, there also have studies about the relationship between coliforms bacteria and enteric virus (Zhang et al., 2012) However, the presence of coliform does not indicate any association with enteric viruses in water This is because coliform and enteric viruses are not homogeneous Viruses are smaller in size than bacteria and survive longer in environments with disinfectants such as chlorine

Some requirements to become microorganism’s indicator of enteric viruses (Grabow, 1986; Kott, 1981) The microorganism’s indicator:

10

- Need to be present in the water where enteric viruses are present

- In terms of quantity, directives are equal to or higher than the number of viruses

- Have resistance in the environment of antiseptic as viral

- Does not easy to grow in water

- Does not cause illness and is proven by easy, quick and cheap methods

- Represented in feces or sewage contamination

1.2 Review on bacteriophages

A bacteriophage is a virus that infects bacteria This is a group of viruses widely distributed in nature, first discovered by British scientist Frederick Twort in 1915, followed by Felix d'Hérelle (1873- 1949) French-Canadian microbiologists have identified parasitic viruses on the bacillus and called them bacteriophages

Phage does not cause a disease to humans but causes a disease to bacteria Each bacterium can be the host of one or more phages Currently, phages were used to study the type of prokaryote, transport of genetic elements in molecular biology studies, treatment of bacterial diseases, epidemiological investigation

Structure and morphology of bacteriophages contain:

- Nucleic acid molecule (genome): most of the bacteriophages, usually with twisted strands of DNA, some with single-stranded DNA, non-DNA-containing phages contain RNA and a sequence of RNA

molecules called capsomer, which is proteins

- Enzymes in the phage's tail contain some enzymes

- Some bacteriophages contain tails and spikes

Phages only infect to consist of bacteria The multiplication of phage in bacteria usually occurs in the following phases:

11

1) Adsorption and penetration stages: In order to penetrate and multiply in the

bacterium, the phage must first find a specific receptor on the surface of the bacterial cell Many studies show that when bacteria mutate, change the surface properties, the phage is not able to penetrate the bacteria Once laced with bacterial cell surface, lysosomes in the phage's tail dissolve the walls of the bacterial cell, then the tail droops the core of the tail into the bacterium, followed by the phage's DNA being injected bacterial cells The capsid shell will stay out of the bacteria

2) Stage of biosynthesis of components: After 2-3 minutes, the phage

deoxyribonuclease (DNase) enzyme breaks down the DNA of the bacterial cell, mRNA and attaches to the enzyme required for the phage synthesized The DNA of the phage formed together with the phage capsid protein is synthesized in the host cell ribosome Enzymes needed for phage synthesis Phage's DNA is formed together with the phage capsid's protein, which is synthesized in the ribosome of the host cell Enzymes needed for phage synthesis Phage's DNA is formed together with the phage capsid's protein, which is synthesized in the ribosome of the host cell

3) Phase assembly and release: DNA components assembled with proteins

form the phage New phages are formed after about 12 minutes and new phage releases usually occur in 25 minutes On average, each bacterium can

be released from 100 to several hundred phages

1.2.1 The value of phages as surrogates for enteric viruses

In the world, the use of bacteriophages as a microbiological indicator for enteric viruses has been investigated by many scientists Several types of bacteriophage have been shown to be able to serve as microorganisms’ indicator for enteric viruses such as somatic coliphages, male-specific F-RNA coliphages, and Bacteroides fragillis phages Somatic coliphages and F-RNA coliphages belong to Coliphages

group, which both infect to the host as E.coli

12

Somatic coliphages are DNA viruses that multiply on the host E coli by attack cell wall of the host Somatic coliphages in river water have a good correlation with enteroviruses (Stetler, 1984) The analysis of somatic coliphages is also quite simple, inexpensive and quickly (Grabow et al., 1998)

Bacteroides fragillis is found in the human intestine (109 -1010/ g faeces) and is associated with enteroviruses and rotaviruses in sewage (Jofre et al., 1989) However, the presence of Bacteroides fragilis phages in human feces is very rare In addition, bacteroids are non-spore with negative gram and are easy to inactivate in environments with high levels of oxygen, so they are also limited to being the indicator of enteric viruses

F-RNA coliphages are the most studied species for use as an indicator of enteric viruses in aquatic environments Because there has the size as small as any enteric virus, while the somatic phage is a bit larger Havelaa et al., 1993 indicates that the association of F-RNA bacteriophages and enteric viruses in river and lake water However, their existence in natural water is still lower than some types of enteric viruses (Schaper et al., 2002) According to Vergara et al., 2015 studies on the relationship between F-RNA coliphages and some enteric viruses such as HAdV, Norovirus noroviruses, RoV rotaviruses in tropical waters

1.2.2 F-specific RNA bacteriophages

F-specific RNA bacteriophages infect to male E.coli through the “pili” and belong

to Leviviridae family This group has linear ssRNA, icosahedral and small These viruses are adsorbed by F-pili to cellular receptors F-RNA infection to the host usually does not occur at temperatures below 30 °C because E coli hosts do not produce F pili at low temperatures and the range of temperatures for 30-37oC (Franke et al., 2005) Host strains used to detect F-specific coliphages in water are

E.coli resistant to streptomycin and ampicillin and Salmonella enterica senovar

Typhimurium WG49 (Stm WG49) Where WG 49 is the most widely used, a salmonella strain possesses F-pilus and antibiotic resistance is used for selective

13

detection of F-phages in the environment F-phage and somatic salmonella phage can infect WG49 However, there are quite a few somatic salmonella phages in the environment and you can judge that all plaques come from F-phages

This group of F-RNAs is divided into 4 groups GI, GII, GIII, and GIV (Vinjé et al., 2004) Including MS-2 in group I, GA in group II, Q in group III, and SP group IV These phages are all present in the disinfection environment, light and the stages of water treatment There have been numerous studies showing that F-specific RNA genotype II and III are found primarily from human waste, while genotypes I and

IV are involved in animal waste (Vinjé et al., 2004)

In the four types of F-RNA coliphages, GI and GII are known to be phages that have a longer lifetime in the environment than GIII and GIV However, their survival depends on factors such as temperature and pH At 4° C, genotypic GI, and GIII F-RNAs are capable of detecting within 110 days, while GIII and GIV will reduce detectability after 3 weeks or 10 days (Long and Sobsey, 2004)

In 2012, the study of Hata et al., (2012) concluded that GI F-RNA phage may be used to represent the minimal and GIII F-RNA phage can be representing to maximal reduction of viruses during wastewater treatment In the research by Haramoto et al., (2015), showed that GI F-RNA coliphages have the potential to be

an indicator of the reduction of viruses through wastewater treatment

1.2.3 Detected method for virus and phages

 Concentrate viruses’ method

Viruses usually appear in small amounts in water, so the virus can be analyzed in a volume of water that needs to be concentrated before analysis

The viral concentrate was studied very early by the use of membranes According to Eisenach et al., 1991, used a membrane for viruses to attach to the membrane for the purpose of determining the size of the virus (Ver et al., 1968) By 1952, ion

14

exchange was used to concentrate polyps (Lo et al., 1952) and Kelly has succeeded

in concentrating viruses from the sewage sewer pattern in 1953

The concentrate viruses’ method base on the adsorption of the particle to the surface

of the membrane which has a charge and elution from that surface by pH-adjusted solution was developed by Wallis and Melnick, 1967 WHO, 1979 introduced a method of virus isolation in wastewater In 1981, the method of concentrate and virus analysis from wastewater was published in the Journal of Water Resources and Sanitation (Greenberg and Taras, 1981)

The size of enteric viruses ranged from 30nm (enterovirus) to 100nm (adenovirus) and have isoelectric points from 2.8 (hepatitis A virus) to 8.0 (rotavirus) (Michen and Graule, 2010) In the water environment, viruses usually have a charge, so they often use a filter to carry the charge to adsorp virus A negative charge membrane is used by many scientists to use and develop Elford, (1931) used pH 4 samples and adsorbed more than 90% of seeded viruses through membranes Recently, Katayama et al., (2002) was developed a procedure for concentration enteric viruses using negatively charged membrane, the recovery yields for poliovirus from 33 to

95 %; Haramoto et al., (2009) was concentrated and recovery NoVs from 250 to 500ml of MilliQ water (186%), tap water (80%), river water (15%) and pond water (39%) by using electronegative membrane combined with aluminum or magnesium

 Culture method

In the 1930s, virologists found that embryonic eggs could be used to cultivate herpes viruses, smallpox, and influenza viruses Although chicken embryos are much simpler in structure than rabbit or rat organisms, chicken embryos remain a complex organ The use of chicken embryos does not completely solve the problems encountered in the cytological effect caused by the virus On the other hand, bacteria also develop well on chicken embryos, on bacterial embryos, it is difficult to accurately assess the effects of the virus

15

Two new findings have led to the development of cell culture methods for virologists and other scientists Firstly, the detection and use of antibiotics to prevent bacterial contamination (Schmidt, 1969) Secondly, biologists have found that proteolytic enzymes, especially trypsin, can separate cells from surrounding tissues without harming them Wash the cells and count them, dilute them and transfer them to plastic bottles, test tubes or boxes The cells in the suspension fluid adsorbed on the surface of the plastic layer, which multiplies and spread to a cellular layer called the monolayer This single cell layer can be subcultured Subculturing is to bring the cultured cells to culture in new culture media Large numbers of subcultures are made up of a single organism template that will be the homogenized cell sample needed for a series of viral effects studies

From the 50s to the 60s of the 20th century more than 400 viruses were isolated and examined In 1950, Enders et al., 1949 was isolated poliovirus For phage, it is possible to use plaque assay to determine the amount of phage present in the sample through spots

 Molecular method

To detect RNA viruses by using animal cell line is difficult and expensive PCR methods are widely used for detection of various microorganisms and it is rapid and sensitive

The polymerase chain reaction (PCR) was discovered by Kary Mullis in 1983 and was awarded the Nobel Prize in Chemistry in eight years later for its enormous applications Thanks to PCR, an inadequate amount of DNA is no longer an obstacle to DNA-based molecular diagnostics and research

According to Mullis, the process by which DNA replicates artificially over many cycles of DNA polymerase replication DNA polymerase is naturally present in living organisms, where it functions as a nucleus of DNA when the cell divides It works by hooking up to the DNA strands and creating complementary fibers According to Mullis's original PCR, the DNA replication enzyme is performed in

16

vitro Double-stranded DNA was split into two strands when heated at 96 ° C However, at this temperature DNA polymerase is destroyed so enzymes are required after each heating period of each cycle Mullis's original PCR procedure is not very effective because it takes a long time, requires a large amount of DNA polymerase, and has to be constantly monitored throughout the PCR process Then, the original procedure was developed using DNA-Polymerase derived from thermophilic bacteria that live in water at temperatures above 110 ° C The DNA polymerase from the organism is thermostable and when used in PCR it is not broken when the mixture is heated to separate the DNA strands Since there is no need to add DNA-polymerase at every cycle, DNA replication can be simpler and more automated

One of the first heat-resistant DNA-polymerases was isolated from Thermus aquaticus and was called Taq Taq polymerase is widely used in PCR experiments (Pavlov et al., 2004)

As already practiced today, PCR needs a lot of ingredients The components are:

1 The template DNA contains the piece of DNA that needs to be amplified

2 Primers, to determine the start and end points of the area to be amplified

3 The DNA-polymerase enzyme catalyzes DNA replication

4 Nucleotides (eg dNTP) are the material for DNA-polymerase to build new DNA

5 A buffer solution, providing a chemical environment for DNA-polymerase PCR is performed during the heat cycle This is a heater that heats and cools in the reaction tube at the correct temperature for each reaction The PCR process consists

of 20 to 30 cycles Each cycle consists of 3 steps:

(1) The temperature rises to 94-96 °C to separate the two strands of DNA This step

is called denaturing, which breaks the hydrogen bond between the two DNA strands Before the first cycle, DNA is usually denatured to the time of opening

(3) Finally, DNA polymerase attaches to the fiber It starts to cling to and work along the DNA strands This step is called stretching The long-term temperature depends on the DNA-polymerase The timing of this step depends on both the DNA-polymerase and the length of the DNA fragment to be amplified 1000bp / 1 minute

Because ribonucleic acid (RNA) cannot play a role as a direct template for PCR, reverse transcription (RT) should be combined with PCR to allow the RNA to be converted to complementary DNA (cDNA) and become template suitable for PCR When the two techniques are combined, this is called reverse transcription polymerase chain reaction (RT-PCR) Previously, three stages of RT-PCR were performed separately, but these three steps are now combined in one trial and are called one-step RT-PCR The use of RT-PCR for the detection of RNA in research and diagnostics provides a fast, effective, sensitive and specific method

Real-time PCR is different from common PCR in that it is capable of detecting and quantifying PCR directly after each cycle of the reaction The implementation of real-time PCR technique is also based on the usual principle of a chain reaction However, the distinctive feature is that the amplified DNA fragment will be detected at each time the reaction takes place (real-time) Real-time PCR consists of two simultaneous processes: DNA replication by PCR and fluorescence imaging proportional to or proportional to the number of DNA fragments

 Advantages and disadvantages

Table 1.1 Advantage and disadvantage of culture and molecular methods

Culture method - Infectivity is determined

- Inexpensive

- 16-24 hours for results

- Cannot detect specific F-RNA group phage

- Sensitive more than culture method

- Qualitative and quantitative

- Analyse each group of RNA phages

determined

- More expensive

1.3 Review on wastewater treatment plant

1.3.1 State of wastewater treatment plant in the world and in Vietnam

Along with the trend of industrial development, the amount of wastewater generated from industrial activities is also diversified Wastewater, if left untreated will make polluted for the world's water resources

19

Nowadays, the world has two trends in waste-water treatment, which are concentrated treatment and decentralized treatment Each trend has its own advantages depending on the region For centralized wastewater treatment models, the advantages are: high processing efficiency should be developed by many developed countries Regarding decentralized wastewater treatment models, the advantages are low investment cost, different treatment technologies and the most appropriate alternative to local conditions in developing countries

According to the Ministry of Natural Resources and Environment (2010), in some urban areas and typical industrial zones in Vietnam, the water source at the place receiving wastewater exceeds the permitted standards according to TSS, BOD5 and COD5 to 10 times the standard allowed, even up to 20 times The content of BOD5and NH4+ is monitored at 1.5-3 times the allowed standard; Coliform numbers in some large breeds also exceeded standards from 1.5 to 6 times

Currently, there are 6 urban cities in Vietnam with 14 wastewater treatment plants Some cities like Nha Trang and Quy Nhon have no concentrated wastewater treatment stations Domestic wastewater is preliminarily treated through septic tanks and then drains and discharged directly into the environment In Hanoi, the Yen So wastewater treatment plant has the largest capacity of 200,000 m3/day invested by Gamuda- Malaysia

Wastewater treatment technologies in Vietnam are mainly different forms of secondary treatment technology by activated sludge, such as conventional activated sludge technology (CAS), anaerobic-deficient aerobic (A2O), oxidative dewatering (OD) and batch biological treatment Activated sludge treatment technology is popular in Jica-funded factories such as Kim Lien, Truc Bach, North Thang Long

1.3.2 Sequencing batch reactor treatment

Previous studies have shown that activated sludge processes also contribute to the elimination of viruses during wastewater treatment According to Ziqiang et al.,

2014 study that activated sludge before disinfection can reduce adenoviruses and

20

enteroviruses by up to 4.1 logs and 3.9 logs, respectively However, the activated sludge process is a process of sludge and water circulating, the virus in the tank can circulate and accumulate over time, so the evaluation of the virus in the activated sludge tank is limited

SBR wastewater treatment technology includes 2 clusters: Selector tank and C - tech tank, Sequencing Batch Reactor (SBR) Biological treatment of wastewater by a continuous batch process This is a form of aerotank The water is taken into the selector tank first and then through the C - tech tank Selector tank will be aeration continuously to facilitate the process of aerobic processing takes place The water is then transferred to the C-tech tank The SBR operates in a cyclic phase with five phases:

- Fill: Wastewater is pumped into the SBR tank for 1-3 hours, in a batch reactor, depending on the treatment target, BOD input level: filling - static, filling - mixing, filling - aerating, creating an atmosphere of aerobic and aerobic in the tank, creating conditions for the microorganism to develop and operate strongly The oxidation of organic compounds takes place in the tank, removing part of BOD or COD in wastewater

- React: Create a biochemical reaction between activated sludge and wastewater by aerobic or ventilate the surface to supply oxygen to water and mix the mixture The duration of this phase is usually about 2 hours, depending on the quality of the wastewater In this phase, nitrification and oxidation of organic matter occur, removal of COD or BOD in water and treatment of nitrogen compounds The nitrification process takes place quickly: ammonium oxidation (NH4+) is carried out by Nitrosomonas bacteria This process converts ammonia to nitrite (NO2-) Other bacteria such as Nitrobacter are responsible for oxidizing nitrite to nitrate (NO3-)

NH4+ +3/2O2 → NO2- + H2O + 2H+

NO2- + 1/2 O2 → NO3-

21

In this stage, it is necessary to control the input parameters such as DO, BOD, COD,

N, P, aeration velocity, temperature, pH, etc so that the activated sludge can be effective for later sedimentation

- Settle: This phase prevents wastewater from entering the SBR tank without blowing and stirring in this phase for the purpose of settling in water in a completely static environment This is also the time of denitrification in the tank with high efficiency It takes about 2 hours The result of this process is

to create two layers in the tank, the upper phase separation layer and the sediment layer being the bottom layer

- Draw: The settled water will be drained by the drainage system without sediment due to the decanter Get rid of water for 0.5 hours

- Idle: Waiting for new batch loading, waiting time depends on the four phases

of operation, the number of tanks and the order of charging water into the tank Discharging of residual sludge is carried out on the last of settling phase if the amount of sludge in the tank is too high This stage is very important in keeping the tank running continuously, partly in the sludge tank, partially circulating in the Selector tank, and the rest in the C - tech tank sedimentation or discharge of water

SBR has been used extensively in countries such as the United States and the United Kingdom for the past two decades, but in Canada, it is still limited because the system requires complete automatic control To overcome this disadvantage, the system has been designed by PLC (Programmable Logic Controller) system, which helps all activities occur precisely and reduce the time as well as operating costs Highlights of SBR technology:

Features in the SBR tank do not need activated sludge circulation The two reaction and settling processes take place in a single tank, activated sludge at the reaction stage and activated sludge recirculation from the settling tank to maintain the concentration Simple and durable structure because operating with an automated