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 ENGINEE[.]
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 i Hanoi, 2019 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 ii ACKNOWLEDGEMENT First of all, I would like to express sincere appreciation and thanks to my research supervisors, Associate Professor Cao The Ha and Professor Hiroyuki Katayama, who kindly support and give guidance to my task This thesis would not be completed without their assistance in every step throughout the process I would like to show gratitude to Associate Professor Ikuro Kasuga, he raised a lot of points in our discussion Without his instructions, the thesis would have been impossible to be done effectively My sincere thanks also goes to Center for Environmental Technology and Sustainable Development – Hanoi University of Sciences, NIHE - Nagasaki Friendship Laboratory, Nagasaki University - Hanoi for supporting and facilitating the student's analysis of samples at the laboratory I would like to thank teachers in Master of Environmental Engineering Program, Vietnam Japan University, their teaching style made an impression on me and I will never forget positive memories of them In short, I would like to thank JICA, Vietnam - Japan University for give me this great opportunity in which I have developed myself A big thank also to my family and my friends, this thesis as a testament to your encouragement and unconditional love I wish to receive the contribution, criticism of the professors Sincerely thank iii TABLE OF CONTENTS 1.1 Swine wastewater and biogas technology in Vietnam 1.1.1 Status of pig farming and swine wastewater in Vietnam 1.1.2 Environmental impact of swine wastewater 1.1.3 Biogas production technology in Vietnam 1.2 Using duckweed pond system for swine wastewater treatment 1.2.1 General information of duckweed 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 iv 3.1 Characteristics of swine wastewater after biogas treatment 33 3.2 Continuous flow treatment system 34 3.2.1 The occurrence of bacterial indicator in continuous flow treatment system 34 3.2.2 The occurrence of Viral indicator and common viruses .35 3.2.3 Positive control 37 3.3 Batch mode system (BMS) 38 3.3.1 The occurrence of bacterial indicator in batch mode system 38 3.3.2 The occurrence of viral indicator in batch mode system .44 3.3.3 Positive control 48 3.4 Other parameters 49 3.4.1 TN, TP in CFS 49 3.4.2 pH, ammonium, Turbidity in CFS and BMS 50 v LIST OF TABLES Table 1.1 The mass of urine and feces excreted daily by a pig 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 Table 1.5 Biogas plant in Vietnam by type of technology Table 1.6 Effectiveness of biogas digester for swine wastewater treatment .7 Table 1.7 The characteristic of Spirodela polyrhiza 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 log concentration (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 Table 3.18 log concentration (mean ± SD) of PMMoV in zone 2,3 of BMS 47 vi 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 log10 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-NH4+ in CFS (mg/L) 52 Figure 3.23 Concentration of N-NH4+ in BMS (mg/L) 52 Figure 3.24 Turbidity in CFS 53 Figure 3.25 Turbidity in BMS 53 vii LIST OF ABBREVIATIONS BMS COD CFU: CFS CETASD DTS E coli MARD CS PCR: PFU RT-PCR QCVN qPCR WW WWT Batch mode system Chemical Oxygen Demand Colony forming unit Continuous flow system Center for Environmental Technology and Sustainable Development Duckweed 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 viii 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 The most commonly known pathogens are bacteria and viruses Many studies have proposed appropriate indicators to identify the presence of pathogens in wastewater The Total coliforms, Escherichia coli have been used as an indicator of fecal pollution and water quality parameters (Pathak et al., 2001) Due to the origin and morphology of FRNA phages similar to enteric viruses, FRNA phages are regarded as viral indicators of water pollution in water environments Currently, researchers have identified common viruses in swine wastewater, including Norovirus GII (NoV GII), hepatitis E virus (HEV), FRNA-GI, etc Because Pepper mild mottle virus (PMMoV) has behaviors similar to enteric viruses, high stability, and abundance in water environments, it can be considered as a viral tracer of fecal pollution (Kitajima et al., 2018) 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: Determine the concentration of pathogens after treatment Assess the fate of pathogens in two different lab-scale systems with continuous flow and batch modes Compare between ponds system with duckweed and pond system without duckweed NoV can survive in the digestive tract They remain infectious after heating to 60°C in 30 minutes Therefore, chlorine-based disinfectants are the most effective for inactivating NoV virus Determination of animal enteric caliciviruses in pigs raises concerns about the possibility of transmission between humans and animals (Mattison et al., 2007) Rotavirus: Rotaviruses is a non-enveloped RNA virus RV is the leading cause of acute gastritis in both humans and pigs RV group A causes diarrhea in piglets Many chemical disinfectants and antiseptics are not effective in inactivating Rotaviruses such as: ether, chloroform, detergents Chemicals such as Phenols, formalin, chlorine, and 95% ethanol have been shown to be more effective UV treatment shows the most effective ability to inactivate Rotaviruses The presence of RV in livestock is a public health problem because it has been detected in human of genotypes of animal strains and vice versa (Straw, 2006) 1.3.2 Microbial indicators a) Bacterial indicators The indicator bacteria are bacteria used to assess the level of fecal pollution of water They are not dangerous to human health and used to indicate the presence of health risks In fecal contains a lot of pathogenic bacteria If eating food containing a large amount of bacteria can cause disease Because of the low concentration of pathogens in the water environment, it is difficult to test them separately So that, the presence of other fecal bacteria more abundant and easily detected such are used as indicators of fecal contamination Coliforms and pathogenic organisms come from the same sources Coliforms are easly identify, often present in larger numbers than dangerous pathogens and react to the environment, WWT like many pathogens Therefore, testing coliform bacteria is reasonable whether other pathogenic organisms are present or not 15 Coliforms bacteria divide into different level (NYSDH, 2017): - Total coliforms indicates the general quality of the supply water about the sanitary condition Total coliformss are bacteria in water, in soil that has been influenced by human or animal waste and surface water - Fecal coliform is the group of the Total coliformss which specifically present in feces of warm-blooded animals Because the specific origins of fecal coliforms so they are more accurate indication than Total coliformss - In the fecal coliform group, Escherichia coli (E coli) is the main species E coli is not grow and reproduce in the environment Therefore, E coli is considered as the best indicator of fecal contamination and the possible presence of pathogenic organisms According to EPA, 1986, the bacterial indicator of fecal contamination need to meet the following criteria: - Whenever enteric pathogens present, microbial should be present - Microbial suitable for all types of water - Microbial must survive longer than the most durable enteric pathogen - Microbial should not grow in the water - Microbial should be present in the intestines of warm-blooded animals b) Viral indicators F-specific RNA bacteriophages (FRNA-phages) Some studies show that Coliform is not always present when enteric viruses are detected (NYSDH, 2017) In that case, F-specific RNA bacteriophages are model organisms suitable to indicate the presence of enteric viruses because they have similar morphology and survival characteristics (Sundram et al., 2002) 16 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 calicivirus family (Henne et al., 2015) MNV have many biological characteristics, including morphology and genetics, replication in the intestine, fecal-oral 17 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) Attaching pathogens to the suspended matter then settle under gravity can remove pathogens from the water column leading to cleaner effluents In duckweed ponds, pathogens can attach to the surface of duckweed and therefore, will be protected from the effects of solar radiation (MacIntyre et al., 2006) Awuah (2006) shows that the reduction of fecal bacteria through attachment to harvested duckweed accounts for less than 1% in the elimination of fecal bacteria 18 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 (