The fate of pathogens in post-biogas swine wastewater treatment using lab-scale duckweed pond.

5 1.2 Using duckweed pond system for swine wastewater treatment .... 9 1.2.3 Using duckweed pond system for swine wastewater treatment .... In this study, the fate of pathogens in post-b

MASTER THESIS ENVIRONMENTAL ENGINEERING

Hanoi, 2019

PROGRAM: ENVIRONMENTAL ENGINEERING

STUDENT ID: 17110041 SUPERVISORS: ASSOC PROF CAO THE HA PROF HIROYUKI KATAYAMA

Hanoi, 2019

iii

ACKNOWLEDGEMENT

First of all, I would like to express sincere appreciation and thanks to my research supervisors, Associate Professor Cao The Ha and Professor Hiroyuki Katayama, who kindly support and give guidance to my task This thesis would not be completed without their assistance in every step throughout the process

I would like to show gratitude to Associate Professor Ikuro Kasuga, he raised a lot of points in our discussion Without his instructions, the thesis would have been impossible to be done effectively

My sincere thanks also goes to Center for Environmental Technology and Sustainable Development – Hanoi University of Sciences, NIHE - Nagasaki Friendship Laboratory, Nagasaki University - Hanoi for supporting and facilitating the student's analysis of samples at the laboratory

I would like to thank teachers in Master of Environmental Engineering Program, Vietnam Japan University, their teaching style made an impression on me and I will never forget positive memories of them In short, I would like to thank JICA, Vietnam

- Japan University for give me this great opportunity in which I have developed myself

A big thank also to my family and my friends, this thesis as a testament to your encouragement and unconditional love

I wish to receive the contribution, criticism of the professors

Sincerely thank

iv

TABLE OF CONTENTS

1.1 Swine wastewater and biogas technology in Vietnam 3

1.1.1 Status of pig farming and swine wastewater in Vietnam 3

1.1.2 Environmental impact of swine wastewater 4

1.1.3 Biogas production technology in Vietnam 5

1.2 Using duckweed pond system for swine wastewater treatment 8

1.2.1 General information of duckweed 8

1.2.2 Factors affecting the growth of duckweed 9

1.2.3 Using duckweed pond system for swine wastewater treatment 10

1.3 Review on pathogens 13

1.3.1 Common pathogens in swine wastewater 13

1.3.2 Microbial indicators 15

1.3.3 Positive control 17

1.3.4 Factors affecting the reduction of the pathogens in the pond system 18

2.1 Swine wastewater and duckweed 20

2.1.1 Swine wastewater 20

2.1.2 Duckweed 21

2.2 Lab-scale duckweed pond 21

2.3 Sample collection in CFS and BMS 25

2.3.1 Water samples 25

2.3.2 Harvesting duckweed 26

2.4 Target parameters analysis 26

2.4.1 Physical - chemical parameters 26

2.4.2 Biological parameters 26

v

3.1 Characteristics of swine wastewater after biogas treatment 33

3.2 Continuous flow treatment system 34

3.2.1 The occurrence of bacterial indicator in continuous flow treatment system 34

3.2.2 The occurrence of Viral indicator and common viruses 35

3.2.3 Positive control 37

3.3 Batch mode system (BMS) 38

3.3.1 The occurrence of bacterial indicator in batch mode system 38

3.3.2 The occurrence of viral indicator in batch mode system 44

3.3.3 Positive control 48

3.4 Other parameters 49

3.4.1 TN, TP in CFS 49

3.4.2 pH, ammonium, Turbidity in CFS and BMS 50

vi

LIST OF TABLES

Table 1.1 The mass of urine and feces excreted daily by a pig 3

Table 1.2 The characteristic of swine wastewater 3

Table 1.3 Some microbiological components in pig waste 4

Table 1.4 Biogas production technology in Vietnam 6

Table 1.5 Biogas plant in Vietnam by type of technology 7

Table 1.6 Effectiveness of biogas digester for swine wastewater treatment 7

Table 1.7 The characteristic of Spirodela polyrhiza 9

Table 1.8 Mean values of various parameters of wastewater and tap water before and after treatment by duckweed 11

Table 1.9 Characteristics of frond biomass of duckweed grown 12

Table 1.10 Bacterial pathogens found in swine wastewater 13

Table 2.1 Preparation of agar for FRNA-phages detection 28

Table 2.2 Components and volumes of RT reaction master mix 31

Table 2.3 RT reaction temperature profile 31

Table 2.4 Components and volumes of q-PCR reaction mixtures 32

Table 2.5 The thermal condition for PMMoV, FRNA-GI 32

Table 2.6 The thermal condition for MNV, NoV GII, HEV 32

Table 3.1 The characteristics of post-biogas swine wastewater 33

Table 3.2 log mean concentration of E coli and Total coliforms (TC) 34

Table 3.3 The mean concentration of FRNA-phages in CFS 35

Table 3.4 log10 concentration of PMMoV in CFS 36

Table 3.5 logconcentration (mean ± SD) of E coli and Total coliforms in zone 2 in BMS 38

Table 3.6 log concentration (mean ± SD) of E coli and Total coliforms

in zone 1 and zone 3 in BMS 38

Table 3.7 The number of E coli taken out in each time sampling 41

Table 3.8 The concentration in different zones of E coli in DTS and CS 42

Table 3.9 The distribution of E coli in DTS and CS 42

Table 3.10 The number of TC taken out in each time sampling 43

Table 3.11 The concentration in different zones of TC in DTS and CS 43

Table 3.12 The distribution of Total coliforms in DTS and CS 43

Table 3.13 The mean concentration of FRNA-phages in water layer of BMS 44

Table 3.14 The number of FRNA-phages taken out in each time sampling 45

Table 3.15 The concentration in different zones of FRNA-phages in DTS and CS 46

Table 3.16 The distribution of FRNA-phages in DTS and CS 46

Table 3.18 log concentration (mean ± SD) of PMMoV in zone 2,3 of BMS 47

vii

LIST OF FIGURES

Figure 1.1 Swine wastewater treatment process 6

Figure 2.1 Pig farm location at Lam Dien - Chuong My - Ha Noi 20

Figure 2.2 Biogas treatment system 20

Figure 2.3 Swine wastewater after biogas treatment 20

Figure 2.4 Duckweed: Spirodela polyrhiza 21

Figure 2.5 Lab-scale continuous flow system 22

Figure 2.6 Lab-scale batch mode system 23

Figure 2.7 Procedure to analyze NoV GII, HEV, MNV, PMMoV, FRNA-GI 29

Fig 3.1 logconcentration of E coli in CFS 34

Fig 3.2 logconcentration of TC in CFS 34

Figure 3.3 log10 concentration of PMMoV in CFS 36

Figure 3.4 The recovery of MNV in CFS 37

Figure 3.5 logconcentration of E coli in BMS 39

Figure 3.6 logconcentration of Total coliforms (TC) in BMS 39

Fig 3.7 Distribution of E coli in DTS (CFU) 42

Fig 3.8 Distribution of E coli in CS (CFU) 42

Figure 3.9 Distribution of TC in DTS (CFU) 44

Figure 3.10 Distribution of TC in CS (CFU) 44

Figure 3.11 log concentration of FRNA-Phages 45

Fig 3.12 Distribution of FRNA-phages in DTS (PFU) 46

Fig 3.13 Distribution of FRNA-phages in CS (PFU) 46

Figure 3.14 log concentration of PMMoV 47

Figure 3.15 The recovery of MNV 48

Figure 3.16 Concentration of TN of CFS (mg/L) 49

Figure 3.17 Concentration of TP of CFS (mg/L) 49

Figure 3.18: Concentration of Photphate 50

Figure 3.20 pH in CFS 51

Figure 3.21 pH in BMS 51

Figure 3.22 Concentration of N-NH4+ in CFS (mg/L) 52

Figure 3.23 Concentration of N-NH4+ in BMS (mg/L) 52

Figure 3.24 Turbidity in CFS 53

Figure 3.25 Turbidity in BMS 53

viii

LIST OF ABBREVIATIONS

BMS Batch mode system

COD Chemical Oxygen Demand

CFU: Colony forming unit

CFS Continuous flow system

CETASD Center for Environmental Technology and Sustainable Development DTS Duckweed treatment system

E coli Escherichia coli

MARD Ministry of Agriculture and Rural Development

CS Control system (no duckweed)

PCR: Polymerase chain reaction

PFU Plaque forming unit

1

INTRODUCTION

Significant of the study

The livestock sector in Vietnam is an integral part of Vietnam's agriculture as well as

an important element of the Vietnamese economy Pig farming accounts for about 60% of the value of the Vietnamese livestock industry, with 27 million pigs, the number one in ASEAN It is estimated that there are 4 million pig farms in the country (MARD, 2017)

The pig production sector in Viet Nam is moving from small household size to intensive farming and large scale In line with that trend, environmental pollution and health risk in pig farms are becoming more serious In the first 8 months of 2018, Vietnam has 8 outbreaks of foot-and-mouth disease and 1 outbreak of porcine reproductive and respiratory syndrome disease (MARD, 2018) Swine wastewater treatments of Vietnam are normally addressed organic matter reduction, but removing pathogens has rarely been considered, which can cause a strong important impact not only on human health but also on the biological safety of pig farms

The most commonly known pathogens are bacteria and viruses Many studies have proposed appropriate indicators to identify the presence of pathogens in wastewater The Total coliforms, Escherichia coli have been used as an indicator of fecal pollution and water quality parameters (Pathak et al., 2001) Due to the origin and morphology

of FRNA phages similar to enteric viruses, FRNA phages are regarded as viral indicators of water pollution in water environments Currently, researchers have identified common viruses in swine wastewater, including Norovirus GII (NoV GII), hepatitis E virus (HEV), FRNA-GI, etc Because Pepper mild mottle virus (PMMoV) has behaviors similar to enteric viruses, high stability, and abundance in water environments, it can be considered as a viral tracer of fecal pollution (Kitajima et al., 2018)

2

Currently, there are many technologies applied in Vietnam to treat swine wastewater, including manure composting, biological agents, biogas, etc The biogas digester is the most popular method However, due to the complex characteristics of swine wastewater, the effluent after biogas treatment still contains high organic, nitrogen compounds, and especially pathogens

It is necessary to find a treatment system to prevent organic, nitrogen pollution, pathogens and minimize the treatment costs The natural system is one of the methods that should be applied after biogas treatment The main advantage is that they consume less power, lower operating and construction costs than standard treatment systems The common plant used in this method is duckweed, due to its rapid growth and high removal of nutrients in wastewater (Ozengin et al., 2007) There have been many studies on the ability of organic matter treatment and nutrients removal by duckweed, but there is not much information about the ability of duckweed to treat pathogens

In this study, the fate of pathogens in post-biogas swine wastewater treatment using duckweed ponds will be investigated

Scope and objectives of the study

This research studied the fate of pathogens after treated by lab-scale duckweed ponds The systems were designed by two parallel lines of ponds, one line contains duckweed, the other line served as a control one There are two different lab-scale systems with continuous flow and batch modes

This study has three main objectives:

1 Determine the concentration of pathogens after treatment

2 Assess the fate of pathogens in two different lab-scale systems with continuous flow and batch modes

3 Compare between ponds system with duckweed and pond system without duckweed

3

LITERATURE REVIEW

1.1 Swine wastewater and biogas technology in Vietnam

1.1.1 Status of pig farming and swine wastewater in Vietnam

Pig farming accounts for about 60% of the value of the Vietnamese livestock industry The number of pigs increased from 27.4 million in 2017 to 28.1 million in 2018 Pork accounts for about 74% of the total meat consumption in Vietnam (TCTK, 2018)

In 2008, small-scale pig farms accounted for 85% of total pigs and 15% of the total

is commercial pig farms (Hoang, 2012) In 2014, 70% of pigs were produced by household farms, the rest were from large-scale commercial pig producers (CCN, 2015) There are 4 million pig farming households in 2014 It is expected that in 2025 this number will decrease by 1.5–2 million households

The transition from traditional pig production to industrial production is creating an increasing amount of pig waste By 2015, pig production has created the highest manure rate (30.3%) (MARD, 2015) Pig manure is not easy to collect because of its slurry form The mass of urine and feces excreted daily by a pig is about 9-11% of its body mass

Table 1.1 The mass of urine and feces excreted daily by a pig

Average weight of Pig

(kg)

Fecal mass (kg/day)

Urine (kg/day)

(Hill el at., 1974) Depending on the method and conditions of livestock production, swine wastewater has different characteristics In swine wastewater, organic compounds account for 70-80% including cellulose, protein, amino acid, fat, carbohydrate, etc, in feces and

4

leftovers Inorganic substances account for 20-30%, the pollution characteristics are shown in detail in the below table:

Table 1.2 The characteristic of swine wastewater

In addition, the feces also contain a variety of bacteria, viruses and parasites In 1kg

of feces contain 2000-5000 eggs of helminths (Nguyen, 2004)

Table 1.3 Some microbiological components in pig waste

of pollution Environmental pollution caused by livestock production is can be the biggest risk to public health

1.1.2 Environmental impact of swine wastewater

Air pollution consists of bad odors emitted from the decomposition process of organic substances in manure, urine, and leftovers The strength of odors depends on the number of excreta discharged, ventilation, temperature, and humidity The

Pigs emit about 70 to 90% of nitrogen, minerals and heavy metals in food Direct discharge of swine wastewater into the soil without treatment causes contamination

of the receiving soil Lands in areas with a high density of pig farms are being polluted

at many levels However, there is still little research and data about this phenomenon (World bank, 2017)

If swine wastewater is not treated well, it will contaminate surface water sources and cause eutrophication The accumulation of pollutants in surface water over a long time may be the cause of the contamination of groundwater due to the permeability process

In terms of microbial contamination, for farm households, the concentration of coliform was 278 times higher than the permissible level (5000 CFU/100 mL) while the industrial farm was 630 times higher than permitted (Phung et al., 2009)

Because of these effects, if swine wastewater is not treat reasonable will greatly affect public health, disease outbreaks in animals, causing serious environmental pollution

1.1.3 Biogas production technology in Vietnam

Currently, pig manure is treated in many ways, including composting, used for biogas and using fresh manure directly as fertilizer In composting, solid waste is collected and mixed to produce organic fertilizer while the liquid is washed away from the floor and discharged into the surrounding environment or fish pond In the biogas method, swine wastewater is collected and processed in biogas digester, gas generated will be

a biogas plant for swine wastewater treatment, while only 12.7% of household farm use it (Dinh, 2009)

According to the report of the Vietnam Institute of Animal Husbandry, swine wastewater treatment is often treated by single method This is a big problem because the effluent not meets discharge standards Most pig farms treat wastewater simply and let the wastewater flow freely into the surrounding environment standards Most pig farms treat wastewater simply and let the wastewater flow freely into the surrounding environment Figure 1.1 shows the swine wastewater treatment process popular in Vietnam:

Figure 1.1 Swine wastewater treatment process

Biogas production technologies in Vietnam are design and processing effect different, biogas technologies applied so far is shown in the table 1.4:

Table 1.4 Biogas production technology in Vietnam

- In addition, the cover is quite heavy, easy to rust

Biogas bags made of

nylon polyethylence

- Easy installation technique Simple operation and less running costs Because of its low price so alot of

households use this type

- The disadvantage is that biogas bags need to avoid sunlight and mechanical damage

Composite biogas

reactors

- Built of hight quality plastic yarn, make molds with high-tech compressors Meet the technical needs, simple design, lightweight, high gas efficiency, easy to install,

(Duong, 2007) The total number of biogas plant installed in Viet Nam, by type of technology and scale, is shown in table 1.5:

Table 1.5 Biogas plant in Vietnam by type of technology

Type of technology used Large – scale farm

MBP: Medium Biogas plants have an average volume of 500m 3 ; LBP: Large Biogas plants have an average volume of 2000 m 3 ; SBP: Small Biogas plants have an average volume of 10m 3

(MARD, 2015) According to the study of Nguyen (2012), the effectiveness of biogas digester for swine wastewater treatment is shown in the table 1.6:

Table 1.6 Effectiveness of biogas digester for swine wastewater treatment

Parameter Influent (mg/L) Effluents (mg/L) Efficiency (%)

3 to 5 times (Vu, 2014) In order to qualify for discharge into the environment, wastewater should be treated further Wastewater treatment using duckweed systems

is considered to be used in the treatment of the organic matter, nutrient and pathogen removal (Smith et al., 2001)

1.2 Using duckweed pond system for swine wastewater treatment

1.2.1 General information of duckweed

Duckweed is divided into four genera including Wolffia, Wolffiella, Spirodela, Lemna belongs to the family of the Lemnaceae, so far, about 40 species are known The fronds of Lemna and Spirodela are oval and flat Wolfia fronds are often crescent- shaped while Wolffiella has a boat shape Spirodela has more than two roots on each frond, Lemna has only one Wolffiella and Wolfia do not have roots (Leng, 2017)

Duckweed is the smallest flowering plant, size from 1mm to 1cm, which floats on the water surface They are dependent on nutrition available in the water (Buijzer et al., 2015)

Lemnacae family appears worldwide, but most in subtropical or tropical areas They

easily grow in the summer season in temperate and cold areas, however, they are very sensitive to frost Duckweed can survive in both fresh and brackish water They do not live in fast flow water, common in still waters, on mud and a rich source of organic nutrients (Borisjuk et al., 2018)

In this study, Spirodela polyrhiza were selected The table 1.7 below shows the

characteristics of this duckweed:

Table 1.7 The characteristic of Spirodela polyrhiza

Active growth period Spring Foliage porosity

After harvest

(USDA, 2015) The duckweed has been showed that very effective in wastewater treatment, due to they grow rapidly and uptake nutrients, particularly phosphate and nitrogen It is also

a source of nutritious food for animals (Ansa et al., 2015), (Chaudhuri et al., 2014)

1.2.2 Factors affecting the growth of duckweed

Duckweed grows in the temperature range of 6-33°C The optimal growth rate when the temperature from 25-31 degrees The treatment efficiency of duckweed is significantly reduced when the temperature is below 17 degrees and above 35 degrees Duckweed can survive in the pH range of 5 to 9 The optimal value is from 6.5 to 7 (Leng, 2017)

Duckweed is very sensitive to the wind, so in very windy regions, wastewater treatment using duckweed pond is not suitable Duckweed will be swept away to the shore of pond by the wind and die, resulting in reduced coverage, enabling algae and mosquitoes to grow (Iqbal, 1999)

10

The natural habitat of Lemnaceae family is quiescent water bodies so they can only withstand water velocity <0.3m/s Fast water flow will limit duckweed cultivation (Leng, 2017)

To grow duckweed requires a stable water source all year round For areas divided into two rainy and dry seasons, it will be difficult to maintain the duckweed pond treatment system The amount of water in the pond decreases in the dry season, if the enffluent is not enough to compensate, the productivity and processing efficiency of the duckweed can be significantly reduced Floods can swamp duckweed away, dilute wastewater in ponds so the content of nutrients decrease may not enough for the development of duckweed (Iqbal, 1999)

1.2.3 Using duckweed pond system for swine wastewater treatment

There are three zones in the duckweed ponds system: aerobic zone, anoxic zone, and anaerobic zone In the aerobic zone, organic matters are oxidized by aerobic bacteria

In the anoxic zone, nitrification and denitrification process take place and constitutes nutrients for the duckweed The organic matter settles to the bottom of the pond, decomposed by anaerobic bacteria producing gases such as CO2, H2S, CH4

- BOD removal: In the duckweed ponds system, because duckweed covers the pond surface so the BOD reduction principle is similar to that of the anaerobic pond (Zirschky et al., 1988)

- Total suspended solids (TSS) removal: The main reason for reducing TSS is due to sedimentation, biodegradation, attach to the root of duckweed and inhibiting algae growth (Iqbal, 1999)

- Nutrient removal: Ammonium and phosphate are important nutrients for the development of duckweed Ammonium reduction is mainly due to ammonium uptake

of duckweed, volatilization of ammonia, sedimentation of organic nitrogen, nitrification and denitrification Phosphate decrease due to plant uptake, adsorption onto organic matter and particles, chemical precipitation (Iqbal, 1999)

Wastewater treatment using duckweed systems have been studied by (Toyama et al., 2018), (Chaudhuri et al., 2014), (Singh et al., 2018), (Yao et al., 2017), (Leng, 2017), etc

According Irfana Showqi et al., (2017), after 15 days of treatment with duckweed, nutrients and heavy metals in tap water and wastewater were significantly reduced

Table 1.8 Mean values of various parameters of wastewater and tap water before

and after treatment by duckweed

(Irfana Showqi et al., 2017)

According to (Sim et al., 2018) indicated that Spirodela polyrhiza was capable of

removing ammonia, nitrate, phosphate, respectively 64%, 30%, 72% and increase 34% their biomass after 12 days

Another study by (X Zhang et al., 2011) showed that Spirodela polyrhiza can survive

in environments with high concentrations of As(V) They have reduced concentration

et al., 2018) The use of swine wastewater after biogas treatment to grow duckweed has a lot of potentials, maximum energy and nutrients can be reused (Iqbal, 1999) The characteristics of frond biomass of duckweed grown in the three effluent samples are shown in table 1.9:

Table 1.9 Characteristics of frond biomass of duckweed grown

in the three effluent samples

Effluent sample and

duckweed species

Carbon content (%)

Nitrogen content (%)

Starch content (%)

Calorific value (MJ/kg) Secondary effluent of municipal wastewater

13

However, the duckweed pond treatment system also has some limitations as follows: The ability to remove pathogens is not clear, if grow duckweed in wastewater they will contain toxic organic compounds and heavy metals, so the use of duckweed to make food for animals cause many risks It not suitable for very windy areas Require

a large land areas for building systems

1.3 Review on pathogens

1.3.1 Common pathogens in swine wastewater

In biology, a pathogen is an infectious microorganism or agent, such as bacterial, viral, fungal, prionic, parasites, algal The most commonly known pathogens in swine wastewater are bacteria and viruses (Ziemer et al., 2010)

a) Bacterial hazards in swine wastewater

Determining the fate of bacterial pathogens from pigs is very difficult The research aspect is mainly the spread of pathogens but still limited More research is needed to determine the factors that affect the survival of pathogens in swine wastewater (USDA, 2006)

The most frequently enteric bacterial pathogens found in swine wastewater are

Escherichia coli, Campylobacter, Salmonella, Enterococcus, Listeria These

pathogens can be transmitted through direct contact with pig manure or through the environment indirectly (Ziemer et al., 2010)

The table 1.10 below summarizes the data on the presence of bacterial pathogens in swine wastewater and show that the results vary widely in the studies, depending on development conditions, pig production systems

Table 1.10 Bacterial pathogens found in swine wastewater

Bacterial pathogens Prevalence

14

: Prevalence = percentage of samples positive for the bacteria : Survival = Length of time (in days) pathogen was detected on the soil or in water.(Ziemer et al., 2010)

b) Common viruses in swine wastewater

Influenza virus: Ifluenza virus is pathogen that can spread easily between humans and

animals The host of the influenza virus consists of humans, marine mammals, bird, pigs, cats and dogs (Webster, 1997) There have been deaths due to swine influenza virus infection (Myers et al., 2006)

Influenza viruses are not labile to the environment outside of the host because they are very sensitive to detergents, heat, lipids solvents, oxidizing agents and irradiation agents It may be inactivated at 56°C for at least 60 minutes or at higher temperature for a shorter time Low pH (pH = 2) also can inactivite influenza viruses (Quinn et al., 2002)

Hepatitis E Virus: HEV is a non-enveloped RNA virus, small and spherical belong

Hepeviridae family Pigs infected with HEV virus are asymptomatic, and some experimental studies show increased liver enzymes in pigs In the United States, HEV virus infection rate is 60-100% Cross species infections between people and pig have been documented (Ziemer et al., 2010)

Little is known about the survival of the HEV virus in the outside of the host Because the HEV virus is spread through the feces-oral so it must be quite stable in the external environment similar to other hepatitis viruses HEV virus is inactivated at a temperature of 71 for at least 20 minutes, easily inactivated by acetic acid and hydroxide sodium and oxidizing agents (Proietto et al., 2016)

Norovirus: NoV belong to Caliciviruses family, small and have surrounding eveloped

RNA NoV virus is now classified into 4 genogroup from GI to GIV NoV GI, GII, GIV infecting humans NoV virus has been found in animals such as pigs, dogs, cattle and mice Pigs infected with NoV GII are a common cause of acute gastroenteritis Porcine strains are genetically and antigenically related to human strains

15

NoV can survive in the digestive tract They remain infectious after heating to 60°C

in 30 minutes Therefore, chlorine-based disinfectants are the most effective for inactivating NoV virus Determination of animal enteric caliciviruses in pigs raises concerns about the possibility of transmission between humans and animals (Mattison et al., 2007)

Rotavirus: Rotaviruses is a non-enveloped RNA virus RV is the leading cause of

acute gastritis in both humans and pigs RV group A causes diarrhea in piglets Many chemical disinfectants and antiseptics are not effective in inactivating Rotaviruses such as: ether, chloroform, detergents Chemicals such as Phenols, formalin, chlorine, and 95% ethanol have been shown to be more effective UV treatment shows the most effective ability to inactivate Rotaviruses

The presence of RV in livestock is a public health problem because it has been detected in human of genotypes of animal strains and vice versa (Straw, 2006)

1.3.2 Microbial indicators

a) Bacterial indicators

The indicator bacteria are bacteria used to assess the level of fecal pollution of water They are not dangerous to human health and used to indicate the presence of health risks In fecal contains a lot of pathogenic bacteria If eating food containing a large amount of bacteria can cause disease Because of the low concentration of pathogens

in the water environment, it is difficult to test them separately So that, the presence

of other fecal bacteria more abundant and easily detected such are used as indicators

of fecal contamination

Coliforms and pathogenic organisms come from the same sources Coliforms are easly identify, often present in larger numbers than dangerous pathogens and react to the environment, WWT like many pathogens Therefore, testing coliform bacteria is reasonable whether other pathogenic organisms are present or not

16

Coliforms bacteria divide into different level (NYSDH, 2017):

- Total coliforms indicates the general quality of the supply water about the sanitary condition Total coliformss are bacteria in water, in soil that has been influenced by human or animal waste and surface water

- Fecal coliform is the group of the Total coliformss which specifically present in feces of warm-blooded animals Because the specific origins of fecal coliforms so they are more accurate indication than Total coliformss

- In the fecal coliform group, Escherichia coli (E coli) is the main species E coli is not grow and reproduce in the environment Therefore, E coli is considered as the

best indicator of fecal contamination and the possible presence of pathogenic organisms

According to EPA, 1986, the bacterial indicator of fecal contamination need to meet the following criteria:

- Whenever enteric pathogens present, microbial should be present

- Microbial suitable for all types of water

- Microbial must survive longer than the most durable enteric pathogen

- Microbial should not grow in the water

- Microbial should be present in the intestines of warm-blooded animals

b) Viral indicators

F-specific RNA bacteriophages (FRNA-phages)

Some studies show that Coliform is not always present when enteric viruses are detected (NYSDH, 2017) In that case, F-specific RNA bacteriophages are model organisms suitable to indicate the presence of enteric viruses because they have similar morphology and survival characteristics (Sundram et al., 2002)

17

FRNA-phages is a virus that infects and replicates within the host cell via the "pili" Salmonella enterica senovar Typhimurium WG49 (Stm WG49) is the most widely used host strain to detect FRNA-phages (Hata et al., 2016)

FRNA-phages have been divided into 4 groups: MS-2 in Group I, GA in Group II,

Qβ in Group III and SP in Group IV (Havelaar & Hogeboom, 1984) Studies have shown that the FRNA-phages group II and group III often involve human waste, while group I and group IV co-related to animal waste such as cow, swine, gull (Hata

et al., 2016) However, the exceptions have been noted FRNA-phages group I was repeatedly discovered in urban wastewater, FNRA-phages group II and III were also detected from animal waste (Stewart et al., 2006) FRNA-phages has been used as one of the rapid screening tests to assess water quality

Pepper mild mottle virus (PMMoV)

Pepper mild mottle virus (PMMoV) has recently been found to be the most abundant

RNA virus in human feces and is a plant virus in the Virgoviridae family

The concentration of PMMoV in human feces from 105 to 1010 (copies/g) (T Zhang

et al., 2005) In Singapore, the US and in Germany, raw sewage also contain PMMoV (Hamza et al., 2011), (Haramoto et al., 2013), (Kuroda et al., 2015) This virus is increasingly considered to be a potential viral indicator for fecal pollution of humans

in water and wastewater treatment systems

Some studies report that elevated PMMoV levels tend to correlate with an increase

in fecal contamination in general, along with the detection of more frequent enteric pathogens PMMoV also exhibits significant stability in water under different environmental conditions (Kitajima et al., 2018)

1.3.3 Positive control

Murine norovirus (MNV) is a small non-enveloped RNA virus belong to the

calicivirus family (Henne et al., 2015) MNV have many biological characteristics, including morphology and genetics, replication in the intestine, fecal-oral

On the other hand, MNV was successfully tested as a positive control process when detecting HAV and NoV in food samples (Karst et al., 2010), (Stals et al., 2011)and HEV in bottled water (Martin et al., 2012)

Prior to virus extraction, MNV was added to samples order to evaluate process efficiencies If the recovery of MNV is greater than 10%, the result is acceptable

1.3.4 Factors affecting the reduction of the pathogens in the pond system

The pathogen removal mechanism is not well understood but it is believed to be mainly through sedimentation and damage by sunlight (Ansa et al., 2015)

Sunlight is a major factor in removal pathogens in pond treatment system (David et al., 2000) The sunlight effect on pathogens depends on the depth of the pond, the shallow ponds have more effective in removing Coliforms (Pearson et al., 2005) Sunlight damages DNA/RNA or the cytoplasmic membrane or depending on their location (Curtis et al., 1992) The effect of sunlight also decreases when light intensity decreases (Van et al., 2000)

Attaching pathogens to the suspended matter then settle under gravity can remove pathogens from the water column leading to cleaner effluents In duckweed ponds, pathogens can attach to the surface of duckweed and therefore, will be protected from the effects of solar radiation (MacIntyre et al., 2006) Awuah (2006) shows that the reduction of fecal bacteria through attachment to harvested duckweed accounts for less than 1% in the elimination of fecal bacteria

19

Temperature is one of the factors promoting the process of mixing ponds (Brissaud

et al., 2003) and may be important in the inactivation of coliforms (Maynard et al., 1999) Total coliforms decay is higher in summer than in winter (Ansa, 2013) Pathogens in water are sensitive to pH changes Curtis et al., 1992 have shown that

both high pH (>8.5) and low pH (<4.0) leads to a higher die-off of E coli, high pH

as an important factor because it not only increased the rate of photo-oxidation but also made the most penetrating wavelength of light bactericidal

According to Klock, 1973, Fecal coliform may survive longer in anaerobic conditions, aeration increases the die-off rate of Fecal coliforms Davies-Colley et al., 1997 found that the inactivation of FRNA viruses increased when increases in

DO levels

The number of pathogens was removed during the wastewater treatment process depends on HRT because it allows more time for the settling of suspended particles that the pathogens were attached Awuah, 2006 have shown that the sedimentation level depends on hydraulic retention time The longer the time, the greater the exposure of pathogens to factors such as sunlight, pH, DO and temperature (Rångeby

et al., 1996)

20

MATERIALS AND METHODS

2.1 Swine wastewater and duckweed

2.1.1 Swine wastewater

Swine wastewater after biogas treatment were collected from a pig farm located at Lam Dien Commune - Chuong My District - Ha Noi The pig farm has a contract with CP Company CP Company will invest in piggy, feeds, veterinary drugs, techniques and consumption of pigs Farmers are responsible for infrastructure and

feeding operation Figure 2.1 shows the location of the pig farm:

Figure 2.1.Pig farm location at Lam Dien - Chuong My - Ha Noi

Figure 2.2 Biogas treatment system Figure 2.3 Swine wastewater after biogas treatment

At the time of sampling, the pig farm was raising 1200 pigs The average consumption

of water per day for cleaning and bathing pigs were approximately 46-49m3 All wastewater and pig manure were collected into biogas digester with volume of

The duckweed Spirodela polyrhiza was taken from a pond at Dinh Cong lake, Hoang

Mai dist., Hanoi City, then it was acclimatized in mini pond containing swine wastewater after biogas treatment with concentration of ammonium of about 40mg/L After adaptation, it was transferred into lab-scale systems located at Environmental Technology Laboratory, Center for Environmental Technology and Sustainable Development (CETASD) and Master of Environmental Engineering (MEE)

Laboratory of Vietnam-Japan University Figure 2.4 shows a picture of duckweed in

a pond:

Figure 2.4 Duckweed: Spirodela polyrhiza

2.2 Lab-scale duckweed pond

There are two different lab-scale systems: one is a continuous flow system (CFS) and the other is a batch mode system (BMS) In every system, there are two lines: Duckweed treatment system (DTS) and control system (CS)

2.2.1 Continous flow system (CFS)

22

The continuous flow systems have two parallel lines of ponds, one line contains duckweed (DTS), the other line was a control system, it has no duckweed (CS) Each line consists of 3 ponds with the same volume of 8 L, a diameter of 2.27 dm The influent’s flow rate was controlled at 0.238 L/h The total hydraulic retention time (HRT) of the system was 100.8h, HRT of each pond was 33.6h

The continuous duckweed pond system was run from 18 March 2019 to 11 April

2019 and located outdoor at CETASD, University of Sciences – VNU, Hanoi This system was exposed to natural conditions and covered by a transparent roof to avoid the impact of rain The temperatures in the range of 19-32 degrees are suitable with duckweed growth conditions

Figure 2.5 Lab-scale continuous flow system Swine wastewater after biogas treatment was settled down for 1 hour then screened through a sieve with mesh opening of 1mm to avoid pump stucking Before flow into the system, the effluent was diluted to adjust the ammonium concentration to about

23

2.2.2 Batch mode system (BMS)

In CFS, because of large dillution and also may be of raw wastewater settling and screening, as described in Chapter 3, the detected pathogen concentrations in ponds system were rather low, making the analysis results unreliable To overcome this problem, a BMS was constructed The dillution rate was also lower to get higher input pathogen concentrations

The batch mode system was located outdoor at MEE laboratory, Vietnam-Japan University Campus and was operated from 28 April to 1 May 2019 The temperatures were in the range of 21-33 °C, this was the same as continously flow system The lab-scale BMS configuration was described in figure 2.6

Figure 2.6 Lab-scale batch mode system

The influent was diluted to adjust the concentration of ammonium to about 70 mg N/L before pouring into the pond system To increase the initial concentration of

target pathogens, E coli, Total coliforms and FRNA-phages were propagated in MEE

24

laboratory and was spiked in each pond Positive control MNV virus were kindly provided by Prof H Katayama (The University of Tokyo)

The volumes of 10 mL FRNA-phages (Concentration = 2.78×1011PFU/mL), 1mL E

coli (Concentration = 5.05×109CFU/mL) and 1mL Total coliforms (Concentration = 5.4×109 CFU/mL) were added into each of 6 ponds Then, the total number of each kind of added pathogen can be calculated by equation:

Total number (PFU or CFU) = Vadded*C Then, each pond has:

Total FRNA-phages = 10 mL×2.78×1011PFU/mL = 27.8×1012PFU

Total E coli = 1 mL×5.05×109CFU/mL = 5.05×109CFU

Total coliforms = 1 mL×5.4×109 CFU/mL = 5.4×109CFU

To follow the fate of pathogens in duckweed ponds the total pond volume was divided into three zones: (i) Zone 1: the surface zone - it is actually duckweed mat, (ii) Zone 2: the middle water zone - it is the most of water volume in the ponds, and (iii) Zone 3: the bottom zone - it is only 1cm layer from the bottom of the pond (V = 0.405L), which contains most of the settled matter during given HRT

(i) Zone 1 (surface zone): To detect microbial amount attached to the surface of duckweed

- After HRT = 81h, all duckweed in the surface of the pond in DTS was harvested by

a 1 mm opening stainless steel sieve into a petri dish (the mass of the clean petri dish:

m1) All the water in the petri dish was decanted and a petri dish containing fresh duckweed was weighed (m2)

- All collected duckweed was mixed with 27 mL LB broth in 15 minutes

- 1mL sample was taken from petri dish above to quantify the concentrations of E

coli, Total coliforms and FRNA-phages (C1)

25

- The concentration of E coli, Total coliforms and FRNA-phages attached on the

duckweed can be calculated as follow:

C =

𝐶1 (𝐶𝐹𝑈𝑚𝐿)×27 (𝑚𝐿)

(ii) Zone 2 (middle water zone): The water of pond except for the bottom layer

- Every 27h, the 50ml syringe was put at a depth of 7cm from the surface of the pond

to collect the sample

- Sample was taken to quantify the concentrations of E coli, Total coliforms, and

Water sample were collected in all ponds Prepare equipment:

- Sterile glass bottle, write information of sample before sampling

- 50 ml syringe

- Styrofoam box contain dry ice pack

Sampling procedure: 150 mL – 550 mL of sample was taken into a glass bottle by 50mL syringe The glass bottle was placed into styrofoam box containing dry ice pack and transfer it to the lab for analysis Storage temperature is 4 degrees

26

2.3.2 Harvesting duckweed

The biomass was harvested manually by a 1 mm opening stainless steel sieve Weight fresh harvested duckweed, then dry the duckweed at 60°C until the weight has no change to get dry weight

2.4 Target parameters analysis

2.4.1 Physical - chemical parameters

pH: In the water, pH effect on microbial activity Measure pH to determine living

environment of microbial species pH was measured with portable sevenCompact

pH meter S220 of METTLER TOLEDO Company

Turbidity: Microorganisms can attach to particles and settle to the bottom, thus need

to determine the turbidity of the water The turbidity was detected by TN-100 water proof Turbidity Meter (Thermo ScientificTM EutechTM)

Temperature: Temperature affects different processes in water and also microbial

activities Temperature was measured via temperature sensor coupled with pH sensor The chemical parameter used to assess water quality in this study is ammonium - N Other chemical parameters were analyzed by other members of research team included: COD, TN, TP

Ammonium - N: The reaction of ammonia, hypochlorite and phenol catalyzed by

nitroprusside produces a dark blue compound Then the intensity of the color was

measured by UNICO spectrophotometer model S2150UV

Procedure: 0.4 mL of phenol solution, 0.4 sodium nitroprusside solution, 1mL oxidizing solution were added into 10mL sample Mix thoroughly after each additional time Avoid samples from light for at least 1 hour at room temperature Then measure the absorbance at 640 nm (APHA, 2005)

2.4.2 Biological parameters

a) E coli and Total coliforms

To calculate how many colonies were present in the original solution, colonies on the plate were counted and then multiply the total dilution factor

Calculated the arithmetic mean of the duplicated colony numbers The number of colonies on plate × dilution factor of sample = colony forming unit (CFU)/mL

b) FRNA-Phages

28

Salmonella Typhymurium WG49, a salmonella strain possess F-pilus and antibiotic

resistance, it is used for selective detection of F-phages in environment F-phage and somatic salmonella phage can infect WG49 (Hata et al., 2016)

- WG49 was provided by Prof H Katayama (The University of Tokyo, Japan)

Table 2.1 Preparation of agar for FRNA-phages detection

Component Unit Liquid media Solid agar