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

quite low because the influent of the system was diluted to meet the concentration of ammonium of around 40 mg N/L. coli and Total coliforms were decreased along the time, but t[r]

i

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

ii

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

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

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

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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

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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 in BMS 38

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

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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 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

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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 RT-PCR

QCVN

Reverse transcriptase polymerase chain reaction National Technical Regulation

qPCR Quantitative polymerase chain reaction

WW Wastewater

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 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 months of 2018, Vietnam has outbreaks of foot-and-mouth disease and 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

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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

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 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)

<10 0,5-1 0,3-0,7

15-45 1-3 0,7-2,0

45-100 3-5 2-4

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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

Parameter Unit Value

Color Pt – Co 350 – 870

Turbidity NTU 420 – 550

BOD5 mg/L 3500 – 8900

COD mg/L 5000 – 12000

SS mg/L 680 – 1200

TP mg/L 36 – 72

TN mg/L 220 – 460

pH - 6.1 – 7.9

(Canh el at.,1998) 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 Unit Value

Coliform MNP/100 g 4×106-108

E Coli MNP/100 g 105-107

Streptococus MNP/100 g 3×102-104

Salmonella Bacteria/25 mL 10-104

Cl Perfrigens Bacterial/mL 10-102

(Nguyen, 2004) Along with the tendency of large-scale pig and intensive farming, environmental pollution is becoming more serious due to poor treating of pig waste and inappropriate use of industrial feed Untreated nutrition, antibiotics, and pathogens in pig manure when discharged into the soil and surrounding water, are observe causes 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

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permissible level of NH3 and H2S are 200 µg/m3 and 42 µg/m3, respectively The

concentration 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 permissible level, respectively (Vu, 2014).A study of environmental pollution caused by livestock in 2009 showed that air pollution (NH3 content) is 18 times higher than the permissible

level for household farms and 21 times for large-scale commercial farms (Phung et al., 2009)

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

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used for cooking and swine wastewater after biogas is used as fertilizer or discharged into fish ponds

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

Type Describes

Biogas digester with fixed cap (KT1- Chinese technology)

- Materials are brick and cement Volume from to 50m3, high durability, gas generated high pressure

- Due to funding from the Netherlands, this type of biogas tunnel is applied in many provinces in Vietnam

7

Floating drum biogas digestor

(KT2-India technology)

- The structure is compact, occupying less construction area, higher cost than other type

- 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 and LBP)

Household scale

(SBP) Total

KT1, KT2 4032 201469 226412

Composite 2390 89147 97320

Other types 8948 159384 141638

Total number of biogas plant 15370 450000 465370

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

(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 (%)

BOD5 1297 ± 201 307 ± 90 76.3 ± 7.1

COD 3022 ± 597 463 ± 127 84.7 ± 5.1

SS 2674 ± 712 373 ± 123 86.1 ± 5.4

VSS 1674 ± 485 244 ± 96 85.4 ± 6.1

TKN 608 ± 87 536 ± 89 11.8 ± 6.0

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farmers to build biogas plants is that they want to reduce odor and fly problems (Luu, 2015) Many farmers 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 are harmful to humans and exceed national standards for wastewater (MARD, 2013) Total coliforms in swine wastewater after biogas treatment exceeds the permitted level (500CFU/100mL) from to 2200 times BOD5 (100mg/L) and COD levels

(300mg/L) from breeding facilities in the north exceed the permitted threshold from to 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 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

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When there are ideal conditions for pH, light, temperature, and nutrients, duckweed can double their biomass in 16h to 48h When meeting unfavorable conditions such as declining temperature, lack of water, duckweed have a special mechanism to survive by flowering in late summer, producing starchy filled structures and sink to the 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 Spring Foliage porosity

summer/winter Porous

After harvest

regrowth rate Slow pH 5.0-8.6

C:N ratio Medium Planting density per 1080-1920

Flower color Green Bloom period Late summer

Foliage color Green Vegetative spread rate Rapid

Genome size(Mbp) 158 Form turions during winter Yes

(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 to The optimal value is from 6.5 to (Leng, 2017)

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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)

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- 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 rarely assessed Studies have not clarified the severity of pathogens to health, although bacteria 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)

Waste water Tap water

Initial Final %

Decrease Initial Final

% Decrease

Nitrogen(N) 7.6 0.5 93.42 0.4 100

Phosphorus(P) 1.1 0.001 99.91 0.11 100

Potassium (K) 18 1.1 93.89 3.8 1.5 60.52

Calcium (Ca) 25.9 0.4 98.46 9.1 0.5 94.51

Magnesium(Mg) 32 2.6 91.88 3.6 0.1 97.22

Copper (Cu) 0.6 85 0.3 100

Zinc (Zn) 0.2 0.01 95 0.1 100

Nickel (Ni) 0.1 0.01 90.00 0

Cadmium (Cd) 0.1 0.001 99.50 0

Chromium (Cr) 0.6 0.001 99.83 0.2 100

Lead (Pb) 0.2 0.01 95.00 0

(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

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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 duckweed species

Carbon content (%)

Nitrogen content (%)

Starch content (%)

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

Spolera polyrhiza 40.3 ± 0.2 5.3 ± 0.2 9.5 ± 0.4 12.1

Lemna minor 40.3 ± 0.2 5.8 ± 0.0 8.2 ± 0.1 12.5

Lemna gibba 39.2 ± 0.0 5.5 ± 0.0 8.7 ± 0.1 11.8

Landoltia punctata 41.1 ± 0.1 5.6 ± 0.2 10.3 ± 0.1 12.9

Secondary effluent of swine wastewater

Spolera polyrhiza 40.5 ± 0.1 6.5 ± 0.0 9.2 ± 0.1 14.1

Lemna minor 40.9 ± 0.3 6.4 ± 0.0 9.4 ± 0.4 14.0

Lemna gibba 40.0 6.2 8.5 11.2

Landoltia punctata 41.8 6.6 8.5 12.8

Secondary effluent of anaerobic digestion

Spolera polyrhiza 43.8 ± 0.2 6.2 ± 0.1 17.3 ± 0.2 15.4

Lemna minor 43.5 ± 0.0 0.1 11.7 ± 0.3 15.4

Lemna gibba 43.0 ± 0.1 6.2 12.9 ± 1.3 15.2

Landoltia punctata 44.0 ± 0.1 6.2 ± 0.2 14.7 ± 0.5 15.2

(Iqbal, 1999)

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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

1 (%) Survival2 (day)

Swine WW Stored waste Plants Soil Water

Salmonella 7.9 – 100 5.2 - 22 16 – 63 16 - 120 35 – 147

Escherichia coili – 22 15.5 – 24 16 – 63 16 – 99 90

Campylobacter 13.5 – 73.9 10.3 16 – 63 – 32 to > 60

Yersinia enterocolitica – 65.4 Unknown 10 – 448

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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 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

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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

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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)

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 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

18

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 is the only type of norovirus that develops efficacy in a small animal host and tissue culture (Henne et al., 2014)

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)

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

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

21

wastewater of 900m3 and volume of HDPE airbag by 600m3 A part of it returned to

the 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 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)

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 ponds with the same volume of 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 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 40 mg N/L

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

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 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

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 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 = mL×5.05×109CFU/mL = 5.05×109CFU

Total coliforms = 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 (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 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

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)

𝑚2−𝑚1 (𝑔) (CFU/g)

(ii) Zone (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 FRNA-phages

(iii) Zone (bottom zone): The volume of water with the high from the bottom of the pond was 1cm, the diameter was 2.27 dm, then V = 0.405L

- After HRT = 81h, a 25mL pipette was used to suck water from the bottom of the pond

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

2.3 Sample collection in CFS and BMS

2.3.1 Water samples

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

26 2.3.2 Harvesting duckweed

The biomass was harvested manually by a 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 hour at room temperature Then measure the absorbance at 640 nm (APHA, 2005)

27

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)

Preparation:

- Chromocult Coliform Agar - LB broth base

- Petri dish

- Screw-top test tube Procedure:

1mL of water sample was added into the first tube containing 9mL of diluting solution The water sample was diluted to 1/10 of initial concentration The serial dilutions were continued until the desired final dilution was achieved Taken mL of the diluted sample from the desired tube to the petri dish Make duplicate plates for each tube Added agar media into petri dishes with diluted samples and mix them 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 and incubated the plates at 37 ± 0.5oC overnight Placed plates upside down to avoid condensed water droplets from falling on the agar surface

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

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)

Preparation: - Petri dish - Glass bottle - Test tube - Solid agar - Liquid media - LB broth base

- 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

Milli-Q mL 500 500

Tryptone g 5

Glucose g 0.5 0.5

NaCl g 4

CaCl2.2H2O (300g/L) mL 0.5 0.5

MgSO4.7H2O (150g/L) mL 0.5 0.5

Bacto agar g - 5.5

Kanamycin (20 g/L) mL - 0.5

Nalidixic Acid (100 g/L) mL - 0.5

Liquid media with well grown WG49 mL - 20

Procedure:

WG49 was added into the liquid media then incubated at 37oC with shaking until

getting turbid The sample was diluted with LB broth appropriately Placed mL of each diluted sample onto the plate Make plates for each dilution factor Mixed the host WG49, Kanamycin and Nalidixic acid with the agar Distributed agar onto the plates and solidify Placed it in face-down manner in an incubator at 37oC overnight

29

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

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

Figure 2.7 below:

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

Preparation:

- 2.5 M MgCl2 solution

- 100 mM (0.2 N) H2SO4 (pH 1.0)

- 100 mM (0.1 N) NaOH (pH 12.8) - 100×TE Buffer solution

- HA filter with pore size: 0.45 μm and diameter: 90 mm - Centriprep YM-50 (Millipore)

Advance Preparation:

- 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 was approximately 10.8 or higher)

- 50 μL of H2SO4 (pH 1.0) and 100 μL of 100× TE Buffer were added to a mL

centrifuge tube The centrifuge tube was used for collecting concentrated solution (alkaline elution solution)

Procedure:

- 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

- mL NaOH (pH 10.8) was applied through the HA filter 5mL tube contains H2SO4

30 and TE Buffer was used to collect elute solution

- The elute solution was added to the outer tube of the Centriprep YM-50 Confirm that the inner lid and outer lid were tightly closed The elute was centrifuged at 2500rpm for 10 minutes, the inner lid and the filtrate were removed

- The outer lid was centrifuged at 2,500 rpm for another 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 Procedure:

- 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 column and was centrifuged at 8000 rpm for The QIAamp spin column was placed into a clean 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 was centrifuged at 8000 rpm for The QIAamp spin column was placed into a clean mL collection tube, the tube containing the filtrated was discarded

31

- 60 µL of buffer AVE was added to the QIAamp Mini spin column and was incubated at room temperature for The QIAamp Mini spin column was centrifuged at 8000 rpm for

- Viral RNA was stored at -20°C  Reverse transcription (RT) Preparation:

- RT reaction master mix - Sterile eppendorf tube - 8-tube trips

- The volume of reagent need for RT step was calculated follow table 2.2:

Table 2.2 Components and volumes of RT reaction master mix

Components Volume for sample (àL)

10ì Reverse transcription buffer

25ì dNTPs 0.8

10× Random primer

MultiScribe TM Reverse transcriptase 1

RNase inhibitor

Molecular grade pure water 3.2

Total 10

Procedure:

- The reagent was thawed after taken out from refrigerator, calculated amount of each reagent was added to the eppendorf tube

- RNA template was mixed with the RT master mix and was distributed into 8-tube

trips

- 8-tube trips were moved to the PCR machine follow temperature in table 2.3:

Table 2.3 RT reaction temperature profile

Step Step Step Step

25°C, 10min 37°C, 120min 85°C, 5min 4°C, ∞

32 Preparation:

- q-PCR reaction mixtures - Sterile eppendorf tube - 96-well plate

- The volume of reagent need for q - PCR step was calculated follow table 2.4

Table 2.4 Components and volumes of q-PCR reaction mixtures

Components Volume (µL)

TaqMan gene Expression master mix 10

Forward primer

Reverse primer

TaqMan Probe 0.5~1.5

}=3 Milli-Q

Sample cDNA

Total 20

Procedure:

- µl cDNA samples with 15 µl qPCR master mix were added in each well of 96 – well plate

- Calibration curve was made from standard plasmid DNA - negative control well was prepared

- The top of 96-well plate was covered by seal, the sealed 96 well plate was centrifuged

- q-PCR (7500 fast real-time PCR) machine was runed follow condition in table 2.5 and table 2.6:

Table 2.5 The thermal condition for PMMoV, FRNA-GI

Two-step cycling Annealing

Step Step Step

95oC (10min) 95oC (15s) 60oC (1min)

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

Two-step cycling Annealing

Step Step Step

33

RESULTS AND DISCUSSION

3.1 Characteristics of swine wastewater after biogas treatment

The following table 3.1 shows the characteristics of the post-biogas swine wastewater of the pig farm in Lam Dien, Chuong My, Hanoi during the study period:

Table 3.1 The characteristics of post-biogas swine wastewater

Parameter Unit Values

QCVN – 62: MT2016/BTNMT1

B column2

pH - 7.97 ± 0.11 5.5-9

TN mg/L 756 ± 345 150

N-NH4+ Mg N/L 472 ± 182 -

TP mg/L 87.3 ± 35.1 -

PO43- mg/L 12 ± 5.6 -

COD mg/L 982 ± 336 300

E coli

(CFU/100 mL)

4.5×104 -

2.48×106 -

Total coliforms (CFU/100 mL) 1.85×10

5 -

4.56×106 5000

1: QCVN = Vietnam Technical Regulation – 62 (Environment) 2016 / (Ministry of Natural Resources and Environment)

2: Column B specifies the value of pollution parameters in livestock wastewater when discharged to water sources not used for domestic water supply purposes

When compared with National Technical Regulation on livestock wastewater (BTNMT, 2016), the pH values were within the permissible range of the standard However, the values of the other parameters exceeded the permitted level to discharge into receiving bodies Specifically, the average concentration of Total coliforms was higher than the permtted level of about 102 - 103 Total Nitrogen was of 756 mg/L, it

was times higher than the permitted level The average COD concentration was of 982 mg/L was 3.2 times higher than the maximum limit

These values of COD and Total coliforms were similar with the values of Việt et al., 2017 (COD = 963 mg/L, Total coliforms = 2.4×105 CFU/100 mL) and Hong, 2012 (Total coliforms = 10.6×106 CFU/100 mL) However, the values of N-NH

34

study were higher than results in the study of Việt et al., 2017 and Hong, 2012 (N-NH4+ = 173 mg N/L, N-NH4+ = 259 mg N/L, respectively), which is because the

farms mays have different sizes and were different in diets and operation conditions

3.2 Continuous flow treatment system

3.2.1 The occurrence of bacterial indicator in continuous flow treatment system During the operation of CFS The sample was collected every 3.5 days in pond containing swine wastewater after dilution, pond and pond with HRT = 0, 33.6 and 100.8h, respectively The analysis results and calculation of E coli and Total coliforms are shown in the table 3.2:

Table 3.2 log mean concentration of E coli and Total coliforms (TC) E coli (CFU/mL)

Logconcentration (mean ± SD)

Total coliformss (CFU/mL)

Logconcentration (mean ± SD)

HRT (h) 33.6 100.8 33.6 100.8

W duckweed

2.51 ± 0.34 0 3.23 ± 0.34 1.03 ± 0.76 0.88 ± 1.01

Wo duckweed 2.51 ± 0.34 0.43 ± 0.86 3.23 ± 0.34 0.68 ± 1.37 0.79 ± 0.91

To make data to be better compared, obtained data from Table 3.2 were converted into a pathogen concentrations profile along the course of wastewater flow in Figure 3.1 and 3.2

Fig 3.1 logconcentration of E coli in CFS Fig 3.2 logconcentration of TC in CFS

Concentration of E coli and Total coliforms in both duckweed treatment system (DTS) and no duckweed control system (CS) at the inlet and at HRT = 100.8 h were

-1

0 33.6 67.2 100.8

log

C

FU

/mL

HRT (h)

w duckweed wo duckweed -1

0 33.6 67.2 100.8

log

C

FU

/mL

HRT (h)

35

quite low because the influent of the system was diluted to meet the concentration of ammonium of around 40 mg N/L In both cases, DTS and CS, E coli and Total coliforms were decreased along the time, but the case of E coli in DTS showed a bit faster reduction, meanwhile, Total coliforms concentration reduction is nearly comparable with a minor advantage of CS

In the influent of the system, the concentration of E coli and Total coliforms were 4.16×102, 2.17×103 CFU/mL, respectively The log reduction of E coli and Total coliforms in both DTS were 2.92 log and 1.87 log, respectively

Although E coli and Total coliforms concentration decreased through the system, the role of the duckweed pond system in reducing bacteria of the experimental system was unclear Therefore, it is necessary to increase the concentration of pathogens by propagated it then spike into the system The further experiment system was presented in section 3.3

3.2.2 The occurrence of Viral indicator and common viruses a) FRNA-phages, HEV, NoV-GII, FRNA-GI

In addition to analyzing bacterial indicator, the viral indicator and other common viruses in swine wastewater are also analyzed The analysis results and calculation of FRNA-phages is shown in the table 3.3:

Table 3.3 The mean concentration of FRNA-phages in CFS

FRNA-phages (PFU/mL)

HRT (h) 33.6h 100.8h

W duckweed ± 1.04 2.21 ± 2.21 0.29 ± 0.39

Wo duckweed ± 1.04 0.57 ± 0.67 0.21 ± 0.39

The concentrations of FRNA-phages detected by plaque assay were also too low, so that the result should not be accepted

HEV, NoV-GII, FRNA-GI were undetermined when detect by q-PCR It is possible to

36

- Due to safety reason, the farm was disinfected every day, therefore the concentration of viruses in swine wasteswater was too low

- The volume of taken samples was not enough (50mL per sample)

b) PMMoV

Concerning PMMoV, one of the most abundant viruses, it gave positive results when detected by q-PCR in 14 of 20 samples The limit of detection (LOD) was in the range of 338 to 1690 PMMoV copies/L The concentration of PMMoV is shown in table 3.4 and figure 3.3 below:

Table 3.4 log10 concentration of PMMoV in CFS

log10 (PMMoV) copies/L Log10 Removal

HRT (h) 33.6 100.8 100.8

W duckweed 4.15 ± 0.31 4.14 ± 0.19 3.601 0.55

Wo duckweed 4.15 ± 0.31 4.00 ± 0.17 3.97± 0.19 0.18

1: Only one sample

Figure 3.3 log10 concentration of PMMoV in CFS

Consider the data from table 3.4 and Figure 3.3, it is clear that the ability to reduce PMMoV of DTS is a bit better than CS by 0.55 log and 0.18 log, respectively In this study, PMMoV decreased after treatment by DTS was lower than results in other

3

0 33.6 67.2 100.8

log

10

cop

ie

s/L

HRT (h)

37

studies The concentration of PMMoV after treatment by an activated sludge process has decreased by 1.7 log and 3.7 log (Hamza et al., 2011), and 0.76 ± 0.53 log (Kitajima et al., 2018)

Although PMMoV is very common in human feces, it was not detected in animal fecal samples such as pigs, cattle, dogs, etc, except chicken and seagulls (Rosario et al., 2009) In our case, the emergence of PMMoV in swine wastewater after biogas shows that the wastewater may be also contaminated with human feces

3.2.3 Positive control

MNV (Murine norovirus) stock solution was provided by Prof Hiroyuki Katayama (The University of Tokyo, Japan)

The recovery efficiency of MNV > 10% is acceptable, which were calculated by the formula (Martin-Latil et al., 2012):

Recovery of MNV = 𝑄𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝑣𝑖𝑟𝑢𝑠 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 𝑎𝑓𝑡𝑒𝑟 𝑠𝑝𝑖𝑘𝑖𝑛𝑔

𝑄𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝑖𝑛𝑜𝑐𝑢𝑙𝑢𝑚 × 100 (%)

The recovery of MNV is shown in figure 3.4:

Figure 3.4 The recovery of MNV in CFS

The results show that the recovery of 19/20 samples was acceptable, ranged from 12% to 49%, this means that the proposed procedure was acceptable

Samples collected from the CFS were subjected to analysis, the results showed that:

19 23 37 22 15 26 20 23 47 28 49 19 27 12 37 15 19 26 21 16 47 43 23 10 20 30 40 50 60

In 33.6 100.8 In 33.6 100.8 In 33.6 100.8 In 33.6 100.8

SET SET SET SET

P erce n tage (% ) HRT (h)

38

- The concentration of pathogen parameters in lines is not much different

- Because of low input concentration values, it is difficult to find the trend of FRNA-phages, E coli, Total coliforms

- Other viruses were undetermined except for PMMoV

Therefore, a further experiment was necessary to make clear the role of duckweed in the fate of pathogens, whether duckweed contributes to reducing microbes or viruses in wastewater through attachment to their surfaces or not

3.3 Batch mode system (BMS)

3.3.1 The occurrence of bacterial indicator in batch mode system

The analysis and calculation of E coli and Total coliforms were conducted for zones: (i) Zone (surface zone: duckweed), (ii) Zone (middle water zone) and (iii) Zone (bottom zone: 1cm from the bottom) Results are shown in table 3.5:

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

E coli (CFU/mL), logconcentration (mean ± SD) log Removal

HRT (h) 27 54 81 81

W duckweed 6.15 ± 0.07 5.87 ± 0.1 5.21 ± 0.14 4.52 ± 0.14 1.62

Wo duckweed 6.08 ± 0.85 5.8 ± 0.1 5.5 ± 0.14 4.89 ± 0.38 1.15

Total coliforms (CFU/mL), logconcentration (mean ± SD) log Removal

HRT (h) 27 54 81 81

W duckweed 6.23 ± 0.04 6.01 ± 0.13 5.34 ± 0.17 4.66 ± 0.14 1.57

Wo duckweed 6.20 ± 0.12 5.94 ± 0.21 5.63 ± 0.06 5.03 ± 0.08 1.18

In the last day of the experiment (HRT = 81h), samples were collected from the surface of DTS and the bottom of all ponds, analysis results may give some image on the distribution of pathogens in duckweed ponds The mean concentrations are shown in the table 3.6 and figure 3.5 - 3.6:

Table 3.6 logconcentration (mean ± SD) of E coli and Total coliforms in zone and zone in BMS

log(E coli) in zone

(CFU/mL)

log(TC) in zone

(CFU/mL)

log(E coli) in zone

(CFU/g)

log(TC) in zone

(CFU/g)

39

W duckweed 5.53 ± 0.17 6.42 ± 0.53 6.43 ± 0.13 7.55 ± 0.14

Wo duckweed 5.93 ± 0.31 6.61 ± 0.38 - -

Figure 3.5 logconcentration of E coli in BMS

Figure 3.6 logconcentration of Total coliforms (TC) in BMS

In zone of the pond, the concentration of E coli and Total coliforms were 2.79×106

CFU/g, 3.74×107 CFU/g, respectively

2 10 12 14 16 2.0 3.0 4.0 5.0 6.0 7.0 8.0

0 27 54 81

log C FU /g log C FU /mL HRT (h)

zone - w duckweed zone - wo duckweed zone - w duckweed zone - wo duckweed zone - w duckweed

2 10 12 14 16

0 27 54 81

log C FU /g log C FU /mL HRT(h)

40

In the zone of the pond, E coli and TC were decreased over time log removal of

E coli and TC in DTS were 1.62log, 1.57 log and 1.15 log, 1.18log in CS,

respectively log removals of DTS is a bit better than CS in reduction of E coli and Total coliformss, 0.47 log and 0.39 log, respectively In the study of Elshafai et al., 2007, TC removal has the logreduction was 4.35 for DTS with HRT = 15 days, in our case HRT ~ 3.4d

In the other hand, E coli and TC also attached to the suspended solid particles then settle to the bottom of the pond, so the concentration of E coli and TC in the bottom layer is higher than in the zone E coli concentration was 3.47×108 and 1.44×108

CFU/mL, and TC was 2.15×109, 1.79×109 CFU/mL, respectively

Compared with CFS, the interpolation values of E coli at HRT = 81h according to graph of E coli concentrationin DTS and CS were 3.53×10-1, 4.33×100 CFU/mL,

respectively log removal of DTS and CS were 3.07 log, 1.98 log, respectively The interpolation values of TC at HRT = 81h according to graph of TC concentrationin DTS and CS were 2.72×101, 5.32×101 CFU/mL, respectively log removal of DTS

and CS were 1.90 log, 1.60 log, respectively Therefore, log removal in both DTS, CS of CFS higher than BMS

From the results of samples and the initial concentration (HRT=0), the distribution of

E coli and Total coliforms between three pond’s zones can be calculated as follow:

Amount of E coli/TC/FRNA-phages (initial)

= (Cz1 × mdw) + (Cz2 × Vz2) + (Cz3 × Vz3) + T + D (CFU or PFU)

Where:

Cz1: Mean concentration of E coli or TC (CFU/g)/or FRNA-phages (PFU/g) in zone (the surface of duckweed), HRT = 81 h

mdw: Mean fresh weigh of duckweed in the surface of pond (5.2812g)

Cz2: Mean concentration of E coli/or TC (CFU/mL)/ or FRNA-phages (PFU/mL) in the zone 2, HRT = 81 h

41

Cz3: Mean concentration of E coli/or TC (CFU/mL)/or FRNA-phages (PFU/mL) in the zone 3, HRT = 81 h

Vz3: Volume of water with the height was cm from the bottom of the pond (mL)

T: The number of E coli/or TC (CFU)/or FRNA-phages (PFU) taken out of sample

collection to analyze (table 3.7, table 3.8)

T = (T0×V0 + T1×V1 + T2×V2 + T3×V3) + (TB×VB)

T0, T1, T2, T3: Mean concentration of E coli/or TC (CFU/mL)/or

FRNA-phages (PFU/mL) in zone in each time of sample collection, duration between samplings = 27h

V0, V1, V2, V3: Volume of water was taken out in zone in each time to analyze

parameters (mL)

TB: Mean concentration of E coli/or TC (CFU/mL)/or FRNA-phages

(PFU/mL) in zone 3, HRT = 81h

VB: Volume of water was taken out in zone (mL)

D: The die off number of E coli/or TC (CFU)/or FRNA-phages (PFU)

In the case of CS, there was no zone

Table 3.7 is shown the number of E coli was taken out to analyze in each time sampling

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

Zones HRT

(h)

Volume (mL)

Concentration of E coli

in DTS (CFU/mL)

The number of

E coli was taken

out in DTS (CFU)

Concentration of E coli in CS

(CFU/mL)

The number of E coli was

taken out in CS (CFU) T0 Zone 150 1.43 × 106 2.15 × 108 1.25 × 106 1.87 × 108 T1 Zone 27 250 7.62 × 105 1.90 × 108 6.77 × 105 1.69 × 108 T2 Zone 54 250 1.70 × 105 4.25 × 107 2.95 × 105 7.38 × 107 T3 Zone 81 550 3.47× 104 1.91 × 107 8.75 × 104 4.81 × 107 TB Zone 81 200 3.57 × 105 7.14 × 107 8.58 × 105 1.72 × 108

Total T - 5.38 × 108 - 6.49 × 108

42

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

HRT (h) Vzone

(mL)

Concentration of

E coli in DTS

(CFU/mL)

Amout of E

coli in DTS

(CFU)

Concentration of E coli in CS

(CFU/mL)

Amout of

E coli in

CS (CFU)

Initial 8000 1.25 × 106 1.14 × 1010 1.25 × 106 9.96 × 109

Zone 11 81 - 2.79 × 106 1.47 × 107 - -

Zone 81 6395 3.47 × 104 2.36 × 108 8.63 × 104 5.52 × 108

Zone 81 405 3.57× 105 1.44 × 108 8.58 × 105 3.47 × 108

Take out2 - - - 5.38 × 108 - 6.49 × 108

Die off3 81 - - 1.05 × 1010 - 8.41 × 109

1: Amout of E coli in Zone = C

z1 (CFU/g) × mdw (g) = CFU

2: Take out = Total T in table 3.7 3: Die off = Initial - (C

z1 × mdw) - (Cz2 × Vz2) - (Cz3 × Vz3) - T

According the above results, the distribution of E coli in the pond can be calculated Table 3.9 is shown the distribution of E coli in DTS and CS

Table 3.9 The distribution of E coli in DTS and CS

Amout of E coli in DTS (CFU)

% Total

Amout of E coli in CS (CFU)

% Total

Initial – Take out 1.09 ×1010 100 9.31 ×109 100

Zone 1.47×107 0.14 - -

Zone 2.22×108 2.03 5.52×108 5.93

Zone 1.44×108 1.33 3.47×108 3.73

Die off 1.05×1010 96.51 8.41×109 90.34

Fig 3.7 Distribution of E coli in DTS (CFU) Fig 3.8 Distribution of E coli in CS (CFU)

Table 3.10 is shown the number of TC was taken out to analyze in each time sampling

0.14 2.03 1.33

96.51

zone zone zone Die off

5.93 3.73

90.34

43

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

Zones HRT

(h) Volume (mL) Concentration of TC in DTS (CFU/mL)

The number of TC was taken

out in DTS (CFU)

Concentration of TC in CS

(CFU/mL)

The number of

TC was taken out in

CS (CFU) T0 Zone 150 1.74 × 106 2.61 × 108 1.63 × 106 2.44 × 108 T1 Zone 27 250 1.08 × 106 2.69 × 108 9.35 × 105 2.34 × 108 T2 Zone 54 250 2.30 × 105 5.76 × 107 4.30 × 105 1.08 × 108 T3 Zone 81 550 4.67 × 104 2.57 × 107 1.08 × 104 5.96 × 107 TB Zone 81 200 4.42 × 106 8.84 × 108 5.36 × 105 1.07 × 109

Total T - 1.50 × 109 - 1.72 × 109

Results of TC in three zones at HRT = 81h were given in table table 3.11 below:

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

HRT (h) Vzone

(mL)

Concentration of TC in DTS

(CFU/mL)

Amout of TC in DTS

(CFU)

Concentration of TC in CS

(CFU/mL)

Amout of TC in CS (CFU)

Initial 8000 1.74 × 106 1.39 × 1010 1.63 × 106 1.30 × 1010

Zone 11 81 - 3.74 × 107 1.97 × 108 - -

Zone 81 6395 4.67 × 104 3.17 × 108 1.08 × 105 7.37 × 108

Zone 81 405 4.42 × 106 1.79 × 109 5.36 × 106 2.17 × 109

Take out2 - - - 1.50 × 109 - 1.72 × 109

Die off3 81 - - 1.01 × 1010 - 8.41 × 109

1: Amout of TC in Zone = C

z1 (CFU/g) × mdw (g) = CFU

2: Take out = Total T in table 3.10 3: Die off = Initial - (C

z1 × mdw) - (Cz2 × Vz2) - (Cz3 × Vz3) - T

According the above results, the distribution of TC in the pond can be calculated Table 3.12 and figure 3.9-3.10 are shown the distribution of TC in DTS and CS

Table 3.12 The distribution of Total coliforms in DTS and CS

Amout of TC in DTS (CFU)

% Total Amout of TC in CS

(CFU)

% Total

Initial – Take out 1.24 ×1010 100 1.13 ×109 100

Zone 1.97 × 108 1.6 - -

Zone 3.17 × 108 2.6 1.97 × 108 6.5

Zone 1.79 × 109 14.4 3.17 × 108 19.2

44

Figure 3.9 Distribution of TC in DTS (CFU) Figure 3.10 Distribution of TC in CS (CFU)

From the distribution of E coli and TC in the different sections of the pond, it is observed that the amount of E coli and TC taken out during multiple sampling after 3.4 days in both systems were similar, by 5.38 × 108 and 6.49 × 108 CFU/mL for E

coli, 1.50 × 109 and 1.72 × 109 CFU/mL for TC, respectively

The distribution of E coli and Total coliforms at DTS in zone were also lower than that of CS The of E coli and Total coliforms attached to the duckweed on the surface was very small, 0.14% and 1.6%, respectively The most obvious thing is that the reduction of E coli and TC was mostly due to the die off

There have not been many studies on the mechanism of the effects of the duckweed on pathogens, therefore more specific studies are needed

3.3.2 The occurrence of viral indicator in batch mode system a) FRNA-phage

In this experiment, FRNA-phages were possitive in all samples The analysis results and calculation of FRNA-phages is show in the table 3.9 and figure 3.11:

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

FRNA-phages (PFU/mL), log concentration (mean ± SD) log

Removal

HRT (h) 27 54 81 81

W duckweed 9.19 ± 0.27 7.85 ± 0.1 7.37 ± 0.14 5.73 ± 0.38 3.4

Wo duckweed 9.12 ± 0.05 8.09 ± 0.14 7.61 ± 0.11 7.11 ± 0.2 1.95

1.6 2.6

14.4

81.5

zone zone zone Die off

6.5 19.2

74.3

45

Figure 3.11 log concentration of FRNA-Phages

FRNA-phages distribution also tends to be similar to E coli and Total coliforms, they were reduced over time

In the surface of duckweed, the concentration of FRNA-phages was 1.12×107 PFU/g This proves that a part of FRNA-phages has attached to duckweed in the surface In the zone of the pond, during operation the system, the logreduction of FRNA-phages in the middle water zone was 3.4 log for DTS, 1.95 log for CS The log reduction of FRNA phages in DTS was higher than the control 1.45 log

FRNA-phages also attached to the suspended solid particles then settle to the bottom of the pond

Table 3.14 is shown the number of FRNA-phages was taken out to analyze in each time sampling

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

Zones HRT

(h) Volume (mL) Concentration of FRNA-phages in DTS (PFU/mL)

The number of FRNA-phages was taken out in

DTS (PFU)

Concentration of FRNA-phages in CS

(PFU/mL)

The number of FRNA-phages was taken out in

CS (PFU) T0 Zone 150 1.80 × 109 2.70 × 1011 1.32 × 109 1.98 × 1011 T1 Zone 27 250 7.28 × 109 1.82 × 1010 1.27 × 108 3.17 × 1010 T2 Zone 54 250 2.43 × 107 6.08 × 109 4.15 × 107 1.04 × 1010 T3 Zone 81 550 7.17 × 105 3.94 × 108 2.02 × 107 1.11 × 1010 TB Zone 81 200 1.47 × 106 2.94 × 108 1.39 × 107 2.78 × 109

Total T - 2.95 × 1011 - 2.53 × 1011

4 10 10

0 27 54 81

lo g P FU/ g log P FU/m L HRT (h)

46

Results of FRNA-phages in three zones at HRT = 81h were given in table table 3.15 below:

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

HRT (h) Vzone

(mL) Concentration of FRNA-phages in DTS (PFU/mL) Amout of FRNA-phages in DTS (PFU) Concentration of FRNA-phages in CS

(PFU/mL)

Amout of FRNA-phages in CS (PFU)

Initial 8000 1.8 × 109 1.44 × 1013 1.32 × 109 1.05 × 1013

Zone 11 81 - 2.12 × 106 1.12 × 109 - -

Zone 81 6395 7.17 × 105 4.87 × 109 2.02 × 107 1.37 × 1011

Zone 81 405 1.47 × 106 5.94 × 108 1.39 × 107 5.62 × 109

Take out2 - - - 2.95 × 1011 - 2.53 × 1011

Die off3 81 - - 1.41 × 1013 - 1.02 × 1013

1: Amout of FRNA-phages in Zone = C

z1 (CFU/g) × mdw (g) = CFU

2: Take out = Total T in table 3.14 3: Die off = Initial - (C

z1 × mdw) - (Cz2 × Vz2) - (Cz3 × Vz3) - T

According the above results, the distribution of FRNA-phages in the pond can be calculated Table 3.16 and figure 3.12-3.13 are shown the distribution of TC in DTS and CS

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

Amout of FRNA-phages in DTS (CFU)

% Total Amout of FRNA-phages

in CS (CFU)

% Total

Initial – Take out 1.41 ×1013 100 1.03 ×1013 100

Zone 1.12×107 0.0001 - -

Zone 4.87×109 0.03 1.37 × 1011 1.33

Zone 5.94×108 0.004 5.62 ×109 0.05

Die off 1.41×1013 99.96 1.02 × 1013 98.61

Fig 3.12 Distribution of FRNA-phages in DTS (PFU) Fig 3.13 Distribution of FRNA-phages in CS (PFU)

0.0001

0.03 0.004

99.96

zone zone zone Die off

1.33 0.05

98.61

47

The amount of FRNA-phages in three zone of the pond in both DTS and CS was very low The amount of FRNA-phages attached to duckweed on the surface very small, the distribution rate was 0.0001% The main cause of reduced FRNA-phages concentration in DTS and CS were due to the die-off

b) PMMoV

In this experiment, PMMoV wasn't spiked into the system The result in table 3.18 is the log concentration of PMMoV available in wastewater, 17/30 samples were positive when detected by q-PCR

Table 3.17 log concentration (mean ± SD) of PMMoV in zone 2,3 of BMS

PMMoV in the middle layer (copies/L), log concentration

(mean ± SD) log removal

PMMoV in zone (copies/L) log concentration

(mean ± SD)

HRT (h) 27 54 81 81 81

W duckweed 3.891 3.87 ±

0.43

3.77 ± 0.24

3.74 ±

0.14 0.15 3.35 ± 0.25

Wo duckweed 4.001 3.931 3.891 3.861 0.14 3.13 ± 0.44

1: Only one sample

log concentration of PMMoV is shown in the figure 3.14:

Figure 3.14 log concentration of PMMoV

3

0 27 54 81

log

c

op

ie

s/L

HRT (h)

48

PMMoV was detected in the samples with the reducing concentrations along the time in DTS was similar with in CS, by 0.15 log and 0.14 log PMMoV PMMoV was decreased over time due to die off and the other factors impacted on them Since the results have not been repeated, the impact of DTS on PMMoV needs further study Compared with CFS, the interpolation values of PMMoV at HRT = 81h according to graph of PMMoV concentrationin DTS and CS were 6.83×103, 9.52×103 copies/L,

respectively log removal of DTS and CS were 0.3 log, 0.2 log, respectively Therefore, log removal of PMMoV in both DTS, CS of CFS higher than BMS 3.3.3 Positive control

MNV virus was added in RNA extraction step The results of recovery of MNV were given in figure 3.15 Fig 3.15 shows that the recovery of 28/30 samples are acceptable, ranged from 11% to 35% MNV is a process control very usefull because it can provide the efficiency of RNA extraction and virus concentration

Figure 3.15 The recovery of MNV

33 30 19 30 24 19 30 18 15 13 19 17 21 21 17 32 25 33 35 11 35 18 21 24 16 24 17 35 10 15 20 25 30 35 40

0 27 54 81 81 108

Zone Zone

Re cov ery o f M N V (% ) HRT (h)

49

3.4 Other parameters

3.4.1 TN, TP in CFS

TN and TP were analyzed during the sampling of the CFS The concentration of Total Nitrogen, Total Phosphorus are shown in figure 3.16 and 3.17, respectively:

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

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

30 50 70 90 110

0 33.6 67.2 100.8

C

on

cen

tr

at

ion

of

TN

(mg

/L)

HRT (h)

w duckweed wo duckweed

0.4 2.4 4.4 6.4 8.4 10.4 12.4

0 33.6 67.2 100.8

C

on

cen

tr

at

ion

of

TP

(mg

/L)

HRT (h)

50

Figure 3.18: Concentration of Photphate

The interpolation values of TN, TP, PO43- at HRT = 81h according to graph of them

concentration in DTS were 49.78 mg/L, 5.621 mg/L, 0.95 mg/L and in CS were 67 mg/L, 8.364 mg/L, 1.21 mg/L, respectively

In the continuous flow system, the DTS was highly capable of removing organic matter as well as nutrients The DTS removal efficiency of, TN, TP, PO43- at HRT =

100.8 were 49%, 48%, 38% respectively This similar to the study of Leng, 2017 (removal efficiency of TN = 50%, TP = 48.4%), but there was no mention about HRT Removal in CS of TN, TP were 30%, 20%, 21%, respectively Therefore, DTS treatment efficiency was higher than CS

3.4.2 pH, ammonium, Turbidity in CFS and BMS a) pH

In BMS, the values of pH at HRT = 81h in DTS and CS were: 7.86 and 8.06, respectively In CFS, the interpolation values of pH at HRT = 81h according to graph of pH values in DTS and CS were 6.73, 7.03, respectively The results are shown in figure 3.20 and figure 3.21

0.6 0.8 1.2 1.4 1.6

0 33.6 67.2 100.8

C

on

cen

tr

aio

n

of

Ph

osp

h

at

e

(mg

/L)

HRT

51

Figure 3.19 pH in CFS Figure 3.20 pH in BMS

Along the time, in both systems, CFS and BMS, the pH decreased gradually It may be due to the nitrification In both systems, DTS had stronger pH reduction as compared with CS This phenomenon may be explained by the existence of duckweed root system, it may serve as a microbial carrying material, then microbial nitrification was stronger than in CS, the consequence was that DTS pH was lower than that of CS

b) NH4+-N:

In BMS, the values of NH4+-N at HRT = 81h in DTS and CS were: 46.89 mg N/L

and 52.19 mg N/L, respectively The removal efficiency of NH4+-N in DTS and CS

at HRT = 81h were 33% and 24%, respectively

In CFS, the interpolation values of NH4+-N at HRT = 81h according to graph of NH4+

-N in DTS and CS were 9.79 mg -N/L and 19.42 mg -N/L, respectively The removal efficiency of NH4+-N in DTS and CS at HRT = 81h were 79% and 59%, respectively

Therefore, the removal efficiency of NH4+-N in CFS higher than BMS

The results are shown in figure 3.20 and figure 3.2

6.0 6.5 7.0 7.5 8.0 8.5

0 33.6 67.2 100.8

pH

HRT (h)

w duckweed wo duckweed

6 6.5 7.5 8.5

0 27 54 81

pH

HRT (h)

52

Fig 3.21 Concentration of N-NH4+ in CFS (mg/L) Fig 3.22 Concentration of N-NH4+ in BMS (mg/L)

Duckweed exposed to both Ammonium-N and Nitrate-N and it preferably uptake for ammonium So that in ponds with duckweed the concentration of ammonium was lower than in ponds without duckweed Then, ammonium reduction in DTS was also higher than the controls in both configurations

c) Turbidity

In BMS, the values of turbidity at HRT = 81h in DTS and CS were: 9.5 NTU and NTU, respectively The removal efficiency of turbidity in DTS and CS at HRT = 81h were 95% and 96%, respectively

In CFS, the interpolation values of turbidity at HRT = 81h according to graph of turbidity in DTS and CS were 2.05 NTU and 3.23 NTU, respectively The removal efficiency of turbidity in DTS and CS at HRT = 81h were 93% and 88%, respectively

0 10 20 30 40 50 60 70 80

0 33.6 67.2 100.8

Co n centratio n o f Amo n iu m (m g/ L) HRT (h)

w duckweed wo duckweed

40 45 50 55 60 65 70 75 80

0 27 54 81

Co n centratio n o f Amm o n iu m (m g/ L) HRT (h)

53

Figure 3.23 Turbidity in CFS Figure 3.24 Turbidity in BMS

Turbidity also decrease steadily Turbidity were similar between two systems This shows that the turbidity decreases mainly due to sedimentation

0 10 20 30 40 50

0 33.6 67.2 100.8

Turb

id

ity

(N

TU)

HRT (h)

w duckweed wo duckweed

0 50 100 150 200 250

0 27 54 81

Turb

id

ity

(N

TU)

HRT(h)

54

CONCLUSION

From results and discussion part, the following conclusions may be made:

1a) For CFS, this study found quantitative values of pathogen parameters for

systems DTS and CS:

E coli, DTS, HRT = 100.8h (81h*) = 5×10-1 (3.53×10-1) CFU/mL

E coli, CS, HRT = 100.8h (81h*) = 5×10-1 (4.33×100) CFU/mL

TC, DTS, HRT = 100.8h (81h*) = 2.91×101 (2.72×101) CFU/mL

TC, CS, HRT = 100.8h (81h*) = 1.89×101 (5.23×101) CFU/mL

PMMoV, DTS, HRT = 100.8h (81h*) = 3.94×103 (6.82×103) copies/L

PMMoV, CS, HRT = 100.8h (81h*) = 9.31×103 (9.52×103) copies/L

FRNA-phages, and other viruses HEV, NoV GII, FRNA-GI were too low to be quantified

1b) For BMS, this study found quantitative values of parameters:

E coli, DTS, zone 1, HRT = 81h: 2.79×106 CFU/g E coli, DTS, zone 2, HRT = 81h: 3.47× 104 CFU/mL E coli, CS, zone 2, HRT = 81h: 8.75 × 104 CFU/mL

E coli, DTS, zone 3, HRT = 81h: 3.57× 105 CFU/mL E coli, CS, zone 3, HRT = 81h: 8.58 × 105 CFU/mL

TC, DTS, zone 1, HRT = 81h: 3.74 × 107 CFU/g TC, DTS, zone 2, HRT = 81h: 4.67 × 104 CFU/mL

TC, CS, zone 2, HRT = 81h: 1.08 × 105 CFU/mL

TC, DTS, zone 3, HRT = 81h: 4.42 × 106 CFU/mL TC, CS, zone 3, HRT = 81h: 5.36 × 106 CFU/mL

FRNA-phages, DTS, zone 1, HRT = 81h: 2.12 × 106 PFU/g FRNA-phages, DTS, zone 2, HRT = 81h: 7.17 × 105 PFU/mL

FRNA-phages, CS, zone 2, HRT = 81h: 2.02 × 107 PFU/mL

55

PMMoV, DTS, zone 2, HRT = 81h: 5.53×103 copies/L

PMMoV, CS, zone 2, HRT = 81h: = 7.24×103 copies/L PMMoV, DTS, zone 3, HRT = 81h: 2.26×103 copies/L

PMMoV, CS, zone 3, HRT = 81h: = 1.36×103 copies/L

The results above show that a part of the microorganism has attached to duckweed on the surface Almost concentration values of E coli, Total coliform, FRNA-phages, PMMoV in DTS lower than in CS

2a) log Removal of 2*2 systems, pathogen parameters in CFS, in BMS were

analyzed:

In CFS, HRT = 81 h

log Removal for E coli: 3.07 log, 1.98 log, in DTS and CS, respectively log Removal for TC: 1.90 log, 1.60 log, in DTS and CS, respectively log Removal for PMMoV: 0.3 log, 0.2 log, in DTS and CS, respectively

In BMS, HRT = 81 h

log Removal for E coli: 1.62 log, 1.15log, in DTS and CS, respectively log Removal for TC: 1.57 log, 1.18 log, in DTS and CS, respectively

log Removal for FRNA-pahes: 3.40 log, 1.15 log, in DTS and CS, respectively log Removal for PMMoV: 0.15 log, 0.14 log, in DTS and CS, respectively

Therefore, the reduction of E coli, Total coliforms, PMMoV in continuous flow system seemed to be higher than batch mode system

2b) Distribution of pathogens in pond depends strongly on conditions in which they

are being treated This study successful in defining the distribution of pathogens in zones of BMS with spiking Quantitative values of pathogen parameters, except the quantity taken off during samplings, are:

In DTS:

E coli were distributed, % = 0.14, 2.03, 1.33, 96.51, in zone 1, 2, 3, and die-off

56

TC were distributed, % = 1.6, 2.6, 14.4, 81.5, in zone 1, 2, 3, and die-off respectively FRNA-phages were distributed, % = 0.0001, 0.03, 0.004, 99.96, in zone 1, 2, 3, and die-off respectively

In CS:

E coli were distributed, % = 5.93, 3.73, 90.34 in zone 2, 3, and die-off respectively

TC were distributed, % = 6.5, 19.2, 74.3, in zone 2, 3, and die-off respectively FRNA-phages were distributed, % = 1.33, 0.05, 98.61, in zone 2, 3, and die-off respectively

3) Other parameters in DTS and CS, HRT = 81h

Log removal of pathogen parameters was presented in 2a)

In CFS:

pH equals to 6.73, 7.03, in DTS and CS, respectively

The removal efficiency of NH4+-N equals to 33% and 24%, respectively

In BMS:

pH equals to 7.86, 8.06, in DTS and CS, respectively

The removal efficiency of NH4+-N equals to 79% and 59%, respectively

57

REFERENCES

1 English

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