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

VIETNAM NATIONAL UNIVERSITY, HANOIVIETNAM-JAPAN UNIVERSITY MA THI TRA MY FATE OF PATHOGENS IN LAB-SCALE DUCKWEED PONDS TREATMENT FOR POST-BIOGAS SWINE WASTEWATER MASTER THESIS ENVIRONMEN

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM-JAPAN UNIVERSITY

MA THI TRA MY

FATE OF PATHOGENS IN LAB-SCALE DUCKWEED PONDS TREATMENT FOR POST-BIOGAS SWINE WASTEWATER

MASTER THESIS ENVIRONMENTAL ENGINEERING

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM-JAPAN UNIVERSITY

MA THI TRA MY

FATE OF PATHOGENS IN LAB-SCALE DUCKWEED PONDS TREATMENT FOR POST-BIOGAS SWINE WASTEWATER

PROGRAM: ENVIRONMENTAL ENGINEERING

STUDENT ID: 17110041

SUPERVISORS: ASSOC PROF CAO THE HA

PROF HIROYUKI KATAYAMA

Hanoi, 2019

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First of all, I would like to express sincere appreciation and thanks to my researchsupervisors, Associate Professor Cao The Ha and Professor Hiroyuki Katayama,who kindly support and give guidance to my task This thesis would not becompleted 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 beenimpossible to be done effectively

My sincere thanks also goes to Center for Environmental Technology andSustainable Development – Hanoi University of Sciences, NIHE - NagasakiFriendship Laboratory, Nagasaki University - Hanoi for supporting and facilitatingthe 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 willnever 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

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

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3.1 Characteristics of swine wastewater after biogas treatment

3.2 Continuous flow treatment system

3.2.1 The occurrence of bacterial indicator in continuous flow treatment system

3.2.2 The occurrence of Viral indicator and common viruses

3.2.3 Positive control

3.3 Batch mode system (BMS)

3.3.1 The occurrence of bacterial indicator in batch mode system

3.3.2 The occurrence of viral indicator in batch mode system

3.3.3 Positive control

3.4 Other parameters

3.4.1 TN, TP in CFS

3.4.2 pH, ammonium, Turbidity in CFS and BMS

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 log 10 concentration of PMMoV in CFS 36

Table 3.5 log concentration (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

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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 log concentration of E coli in CFS 34

Fig 3.2 log concentration of TC in CFS 34

Figure 3.3 log 10 concentration of PMMoV in CFS 36

Figure 3.4 The recovery of MNV in CFS 37

Figure 3.5 log concentration of E coli in BMS 39

Figure 3.6 log concentration 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-NH 4+ in CFS (mg/L) 52

Figure 3.23 Concentration of N-NH 4+ in BMS (mg/L) 52

Figure 3.24 Turbidity in CFS 53

Figure 3.25 Turbidity in BMS 53

Batch mode system

Chemical Oxygen Demand

Colony forming unit

Continuous flow system

Center for Environmental Technology and Sustainable DevelopmentDuckweed treatment system

Escherichia coli

Ministry of Agriculture and Rural Development

Control system (no duckweed)

Polymerase chain reaction

Plaque forming unit

Reverse transcriptase polymerase chain reaction

National Technical Regulation

Quantitative polymerase chain reaction

Wastewater

Wastwater treatment

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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 about60% of the value of the Vietnamese livestock industry, with 27 million pigs, thenumber one in ASEAN It is estimated that there are 4 million pig farms in thecountry (MARD, 2017)

The pig production sector in Viet Nam is moving from small household size tointensive farming and large scale In line with that trend, environmental pollutionand 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 porcinereproductive and respiratory syndrome disease (MARD, 2018) Swine wastewatertreatments of Vietnam are normally addressed organic matter reduction, butremoving pathogens has rarely been considered, which can cause a strong importantimpact 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 haveproposed appropriate indicators to identify the presence of pathogens in wastewater.The Total coliforms, Escherichia coli have been used as an indicator of fecalpollution and water quality parameters (Pathak et al., 2001) Due to the origin andmorphology of FRNA phages similar to enteric viruses, FRNA phages are regarded

as viral indicators of water pollution in water environments Currently, researchershave identified common viruses in swine wastewater, including Norovirus GII(NoV GII), hepatitis E virus (HEV), FRNA-GI, etc Because Pepper mild mottlevirus (PMMoV) has behaviors similar to enteric viruses, high stability, andabundance in water environments, it can be considered as a viral tracer of fecalpollution (Kitajima et al., 2018)

Currently, there are many technologies applied in Vietnam to treat swinewastewater, including manure composting, biological agents, biogas, etc Thebiogas digester is the most popular method However, due to the complexcharacteristics of swine wastewater, the effluent after biogas treatment still containshigh 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 themethods that should be applied after biogas treatment The main advantage is thatthey consume less power, lower operating and construction costs than standardtreatment systems The common plant used in this method is duckweed, due to itsrapid growth and high removal of nutrients in wastewater (Ozengin et al., 2007).There have been many studies on the ability of organic matter treatment andnutrients 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 usingduckweed ponds will be investigated

Scope and objectives of the study

This research studied the fate of pathogens after treated by lab-scale duckweedponds The systems were designed by two parallel lines of ponds, one line containsduckweed, the other line served as a control one There are two different lab-scalesystems 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

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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 Porkaccounts 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 byhousehold 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 anincreasing amount of pig waste By 2015, pig production has created the highestmanure rate (30.3%) (MARD, 2015) Pig manure is not easy to collect because ofits 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

(Hill el at., 1974)

Depending on the method and conditions of livestock production, swine wastewaterhas different characteristics In swine wastewater, organic compounds account for70-80% including cellulose, protein, amino acid, fat, carbohydrate, etc, in feces and

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

Table 1.2 The characteristic of swine wastewater Parameter

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 Parameters

in pig manure when discharged into the soil and surrounding water, are observecauses 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 oforganic substances in manure, urine, and leftovers The strength of odors depends

on the number of excreta discharged, ventilation, temperature, and humidity The

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permissible level of NH3 and H2S are 200 µg/m3 and 42 µg/m3, respectively Theconcentration of NH3 and H2S in air emitted from pig farm in the North of Vietnam

is reported to be 7–18 times higher and 5–50 times higher than the permissiblelevel, respectively (Vu, 2014) A study of environmental pollution caused bylivestock in 2009 showed that air pollution (NH3 content) is 18 times higher than thepermissible level for household farms and 21 times for large-scale commercialfarms (Phung et al., 2009)

Pigs emit about 70 to 90% of nitrogen, minerals and heavy metals in food Directdischarge 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 beingpolluted at many levels However, there is still little research and data about thisphenomenon (World bank, 2017)

If swine wastewater is not treated well, it will contaminate surface water sourcesand cause eutrophication The accumulation of pollutants in surface water over along time may be the cause of the contamination of groundwater due to thepermeability process

In terms of microbial contamination, for farm households, the concentration ofcoliform was 278 times higher than the permissible level (5000 CFU/100 mL) whilethe 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 affectpublic 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 biogasand using fresh manure directly as fertilizer In composting, solid waste is collected andmixed to produce organic fertilizer while the liquid is washed away from the floor anddischarged into the surrounding environment or fish pond In the biogas method, swinewastewater is collected and processed in biogas digester, gas generated will be

used for cooking and swine wastewater after biogas is used as fertilizer ordischarged into fish ponds.

In pig production, the use of biogas digesters for swine wastewater treatment isrelatively widespread About 53% of industrial farms in the south, 60% in the northand 42% in the central region are reported to have used biogas digesters for swinewastewater treatment (Vu, 2014) The majority of the commercial farms (81%) have

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

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

Pigfarm Biogas Stabiliza Draina

Figure 1.1 Swine wastewater treatment process

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

Table 1.4 Biogas production technology in Vietnam Type

Biogas digester with

fixed cap

(KT1-Chinese technology)

biogas tunnel is applied in many provinces in Vietnam

6

Floating drum biogas

Total number of biogas plant

MBP: Medium Biogas plants have an average volume of 500m3; LBP: Large Biogas plants have an average volume of 2000 m3; SBP: Small Biogas plants have an average volume of 10m3

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farmers to build biogas plants is that they want to reduce odor and fly problems (Luu,2015) Many farmers do not know that wastewater after biogas treatment is not safe.

In fact, the biogas plant only reduces the concentration of E coli by 2log10

CFU/mL The biogas digester sample is still positive for some pathogens that areharmful to humans and exceed national standards for wastewater (MARD, 2013).Total coliforms in swine wastewater after biogas treatment exceeds the permittedlevel (500CFU/100mL) from 4 to 2200 times BOD5 (100mg/L) and COD levels(300mg/L) from breeding facilities in the north exceed the permitted threshold from

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 andpathogen 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 onthe water surface They are dependent on nutrition available in the water (Buijzer etal., 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 arevery 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 oforganic nutrients (Borisjuk et al., 2018)

When there are ideal conditions for pH, light, temperature, and nutrients, duckweedcan double their biomass in 16h to 48h When meeting unfavorable conditions such

as declining temperature, lack of water, duckweed have a special mechanism tosurvive by flowering in late summer, producing starchy filled structures and sink tothe bottom during the winter until favorable conditions available (Leng, 2017)

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

1.2.2 Factors affecting the growth of duckweed

Duckweed grows in the temperature range of 6-33°C The optimal growth rate whenthe temperature from 25-31 degrees The treatment efficiency of duckweed issignificantly reduced when the temperature is below 17 degrees and above 35degrees Duckweed can survive in the pH range of 5 to 9 The optimal value is from6.5 to 7 (Leng, 2017)

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

The natural habitat of Lemnaceae family is quiescent water bodies so they can onlywithstand 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 dividedinto two rainy and dry seasons, it will be difficult to maintain the duckweed pondtreatment system The amount of water in the pond decreases in the dry season, ifthe enffluent is not enough to compensate, the productivity and processingefficiency of the duckweed can be significantly reduced Floods can swampduckweed away, dilute wastewater in ponds so the content of nutrients decrease maynot 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, andanaerobic zone In the aerobic zone, organic matters are oxidized by aerobic bacteria Inthe anoxic zone, nitrification and denitrification process take place and constitutesnutrients 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 pondsurface 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 tosedimentation, biodegradation, attach to the root of duckweed and inhibiting algaegrowth (Iqbal, 1999)

- Nutrient removal: Ammonium and phosphate are important nutrients for thedevelopment of duckweed Ammonium reduction is mainly due to ammoniumuptake of duckweed, volatilization of ammonia, sedimentation of organic nitrogen,nitrification and denitrification Phosphate decrease due to plant uptake, adsorptiononto organic matter and particles, chemical precipitation (Iqbal, 1999)

- Heavy metal removal: Heavy metals can be decrease by sedimentation and plant uptake (Iqbal, 1999).

The risk of pathogens in the duckweed ponds treatment system has been rarelyassessed Studies have not clarified the severity of pathogens to health, althoughbacteria tend to accumulate on the surface of duckweed

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 Parameters (mg/L)

(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 increase34% 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

of As in contaminated areas within 72 hours, it accumulated 999 ± 95 mg As/kg duckweed.

The results of Falabi et al., (2002) determined the reduction efficiency of duckweed pond for Total coliforms by 61%, fecal coliforms by 62% and coliphage by 40% Duckweed is raised in swine wastewater and anaerobic contain Carbon, Nitrogen, starch and higher calorific value than duckweed raised in urban wastewater

(Toyama 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

However, the duckweed pond treatment system also has some limitations asfollows: The ability to remove pathogens is not clear, if grow duckweed inwastewater 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 verywindy 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 inswine 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 researchaspect is mainly the spread of pathogens but still limited More research is needed todetermine 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 theenvironment indirectly (Ziemer et al., 2010)

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

Table 1.10 Bacterial pathogens found in swine wastewater Bacterial pathogens

1 : Prevalence = percentage of samples positive for the bacteria 2: 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 swineinfluenza virus infection (Myers et al., 2006)

Influenza viruses are not labile to the environment outside of the host because theyare very sensitive to detergents, heat, lipids solvents, oxidizing agents andirradiation agents It may be inactivated at 56°C for at least 60 minutes or at highertemperature for a shorter time Low pH (pH = 2) also can inactivite influenzaviruses (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 someexperimental studies show increased liver enzymes in pigs In the United States,HEV virus infection rate is 60-100% Cross species infections between people andpig 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 inthe 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 andhydroxide 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, GIVinfecting humans NoV virus has been found in animals such as pigs, dogs, cattle andmice Pigs infected with NoV GII are a common cause of acute gastroenteritis Porcinestrains are genetically and antigenically related to human strains

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 forinactivating NoV virus Determination of animal enteric caliciviruses in pigs raisesconcerns 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 inactivatingRotaviruses such as: ether, chloroform, detergents Chemicals such as Phenols,formalin, chlorine, and 95% ethanol have been shown to be more effective UVtreatment shows the most effective ability to inactivate Rotaviruses

The presence of RV in livestock is a public health problem because it has beendetected 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 ofwater They are not dangerous to human health and used to indicate the presence ofhealth 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 ofpathogens 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 areeasly 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

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

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

- Fecal coliform is the group of the Total coliformss which specifically present infeces of warm-blooded animals Because the specific origins of fecal coliforms sothey 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 pathogenicorganisms

According to EPA, 1986, the bacterial indicator of fecal contamination need to meetthe 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 aredetected (NYSDH, 2017) In that case, F-specific RNA bacteriophages are modelorganisms suitable to indicate the presence of enteric viruses because they havesimilar morphology and survival characteristics (Sundram et al., 2002)

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 widelyused 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 haveshown 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 Iwas repeatedly discovered in urban wastewater, FNRA-phages group II and III werealso 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 containPMMoV (Hamza et al., 2011), (Haramoto et al., 2013), (Kuroda et al., 2015) Thisvirus is increasingly considered to be a potential viral indicator for fecal pollution ofhumans 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 entericpathogens PMMoV also exhibits significant stability in water under differentenvironmental 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

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transmission, are similar between norovirus in humans and murine (Hwang et al.,2014).

MNV is very abundant in research mice and is also found in wild rodents This isthe only type of norovirus that develops efficacy in a small animal host and tissueculture (Henne et al., 2014)

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

Prior to virus extraction, MNV was added to samples order to evaluate processefficiencies 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 bemainly through sedimentation and damage by sunlight (Ansa et al., 2015)

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

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

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 butalso made the most penetrating wavelength of light bactericidal

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

DO levels

The number of pathogens was removed during the wastewater treatment processdepends on HRT because it allows more time for the settling of suspended particlesthat the pathogens were attached Awuah, 2006 have shown that the sedimentationlevel depends on hydraulic retention time The longer the time, the greater theexposure of pathogens to factors such as sunlight, pH, DO and temperature(Rångeby et al., 1996)

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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 atLam Dien Commune - Chuong My District - Ha Noi The pig farm has a contractwith 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 Allwastewater and pig manure were collected into biogas digester with volume of

wastewater of 900m3 and volume of HDPE airbag by 600m3 A part of it returned tothe fish pond and the other part was discharged directly into the environment after

storage in a stabilization pond Figure 2.2 shows the biogas treatment system of pig farm and figure 2.3 shows the effluent discharge well from biogas digester.

2.1.2 Duckweed

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 containingswine wastewater after biogas treatment with concentration of ammonium of about40mg/L After adaptation, it was transferred into lab-scale systems located atEnvironmental Technology Laboratory, Center for Environmental Technology andSustainable Development (CETASD) and Master of Environmental Engineering

(MEE) Laboratory of Vietnam-Japan University Figure 2.4 shows a picture of

2.2.1 Continous flow system (CFS)

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The continuous flow systems have two parallel lines of ponds, one line containsduckweed (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 retentiontime (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 Thissystem was exposed to natural conditions and covered by a transparent roof to avoidthe impact of rain The temperatures in the range of 19-32 degrees are suitable withduckweed growth conditions

Figure 2.5 Lab-scale continuous flow systemSwine wastewater after biogas treatment was settled down for 1 hour then screenedthrough a sieve with mesh opening of 1mm to avoid pump stucking Before flowinto the system, the effluent was diluted to adjust the ammonium concentration toabout 40 mg N/L

-At the start the system, duckweed was placed in half of the surface of each pond inDTS

-Every 3.5 days (84 h), the duckweed biomass increased was harvested Then weigh the harvested duckweed, analyze the water parameters in each pond

2.2.2 Batch mode system (BMS)

In CFS, because of large dillution and also may be of raw wastewater settling andscreening, as described in Chapter 3, the detected pathogen concentrations in pondssystem were rather low, making the analysis results unreliable To overcome thisproblem, a BMS was constructed The dillution rate was also lower to get higherinput pathogen concentrations

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

Figure 2.6 Lab-scale batch mode systemThe influent was diluted to adjust the concentration of ammonium to about 70 mg N/Lbefore pouring into the pond system To increase the initial concentration of target

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

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

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

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

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

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

Total E coli = 1 mL×5.05×109 CFU/mL = 5.05×109 CFUTotal coliforms = 1 mL×5.4×109 CFU/mL = 5.4×109 CFU

To follow the fate of pathogens in duckweed ponds the total pond volume was dividedinto 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 petridish: m1) All the water in the petri dish was decanted and a petri dish containingfresh 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)

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

duckweed can be calculated as follow:

C =(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 by50mL syringe The glass bottle was placed into styrofoam box containing dry icepack and transfer it to the lab for analysis Storage temperature is 4 degrees

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 weighthas 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 teamincluded: 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 wasmeasured by UNICO spectrophotometer model S2150UV

Procedure: 0.4 mL of phenol solution, 0.4 sodium nitroprusside solution, 1mLoxidizing solution were added into 10mL sample Mix thoroughly after eachadditional 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

E coli and Total coliformss were detected by Chromocult® Coliform Agar using pour

plate method The Salmon-Gal substrate in Chromocult® Coliform Agar reacts with

β– D – galactosidase of coliform and results in red colonies On the other hand,both the X – glucuronide substrate and the Salmon – Gal substrate in Chromocult®

Coliform Agar react with β – D – glucuronidase of E coli and results in blue or purple colonies By this way, E coli and Total coliforms can be detected

simultaneously ( Hata et al., 2016)

of the diluted sample from the desired tube to the petri dish Make duplicate platesfor each tube Added agar media into petri dishes with diluted samples and mixthem thoroughly but gently not to spill the agar solution from the bottom plates.Each plate approximately requires 20 mL of agar media Solidify the agar andincubated the plates at 37 ± 0.5oC overnight Placed plates upside down to avoidcondensed water droplets from falling on the agar surface

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

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

b) FRNA-Phages

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

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

c) NoV GII, HEV, MNV, PMMoV, FRNA-GI

Common viruses were detected by quantitative-PCR following the procedure as in

Figure 2.7 below:

Concentration RNA extraction RT q - PCR

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

- 100×TE Buffer solution

- HA filter with pore size: 0.45 μm and diameter: 90 mm

- Centriprep YM-50 (Millipore)

- 1 mL of H2SO4 (pH 1.0) was added to 200 mL of MilliQ

-NaOH (pH 12.8) was diluted 100 times by MilliQ water (confirm that the pH wasapproximately 10.8 or higher)

-50 μL of H2SO4 (pH 1.0) and 100 μL of 100× TE Buffer were added to a 5 mL centrifuge tube The centrifuge tube was used for collecting concentrated solution(alkaline elution solution)

- 2.5M MgCl2 solution was added to the sample follow the ratio 1:100

- Syringe was used to apply the sample through the HA filter

- 200 mL acid solution was applied through the HA filter

- 5 mL NaOH (pH 10.8) was applied through the HA filter 5mL tube contains

H2SO4

and TE Buffer was used to collect elute solution.

- The elute solution was added to the outer tube of the Centriprep YM-50 Confirmthat the inner lid and outer lid were tightly closed The elute was centrifuged at2500rpm for 10 minutes, the inner lid and the filtrate were removed

- The outer lid was centrifuged at 2,500 rpm for another 5 minutes, and the filtrate was removed as written above

-The solution was collected in the Centriprep Pipette was used to measure volume The solution was stored at -20°C before going to qPCR detection

RNA extraction

Preparation:

- QIAamp Viral RNA Mini Kit

- 1.5 mL sterilized tube

- 2 µL MNV virus and 140 µL sample was added into 1.5 mL tube

- 560 µL of prepared buffer AVL contatining carrier RNA was added into 1.5 mL above then incubated at room temperature for 10 minute

- 560 µL of ethanol (99.7%) was added to the sample, the tube was mixed and centrifuged

-630 µL of solution from the above step was added to the QIAamp Mini spin columnand was centrifuged at 8000 rpm for 1 min The QIAamp spin column was placed into

a clean 2 mL collection tube, the tube containing the filtrated was discarded

- The above step was repeated

- 500 µL Buffer AW1 was added to the QIAamp Mini spin column and wascentrifuged at 8000 rpm for 1 min The QIAamp spin column was placed into aclean 2 mL collection tube, the tube containing the filtrated was discarded

- 500 µL Buffer AW2 was added to the QIAamp Mini spin column and wascentrifuged at 14000 rpm for 3 min The QIAamp spin column was placed into aclean 1.5 mL collection tube, the tube containing the filtrated was discarded