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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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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) LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Concentraion of Phosphate (mg/L) 1.6 1.4 1.2 0.8 0.6 33.6 67.2 100.8 HRT w duckweed wo duckweed 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 50 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 8.5 8.0 7.5 7.5 pH pH 8.5 7.0 6.5 6.5 6.0 33.6 67.2 100.8 HRT (h) w duckweed wo duckweed Figure 3.19 pH in CFS 27 54 81 HRT (h) w duckweed wo duckweed 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 51 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 33.6 67.2 100.8 HRT (h) w duckweed wo duckweed Fig 3.21 Concentration of N-NH4+ in CFS (mg/L) Concentration of Ammonium (mg/L) Concentration of Amonium (mg/L) 80 70 60 50 40 30 20 10 80 75 70 65 60 55 50 45 40 27 w duckweed 54 81 HRT (h) wo duckweed 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 52 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 250 40 200 Turbidity (NTU) Turbidity (NTU) 50 150 30 100 20 10 50 0 0 33.6 67.2 100.8 HRT (h) w duckweed wo duckweed 27 w duckweed Figure 3.23 Turbidity in CFS 54 81 HRT(h) wo duckweed 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 53 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 FRNA-phages, DTS, zone 3, HRT = 81h: 1.47 × 106 PFU/mL FRNA-phages, CS, zone 3, HRT = 81h: 1.39 × 107 PFU/mL 54 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 respectively 55 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 The chemical parameters and physical parameters of both systems were significantly reduced 56 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com REFERENCES English Ansa, E D (2013) The removal of faecal coliforms in waste stabilization pond systems and eutrophic lakes IHE Delft Institute for Water Education Ansa, E D O., Awuah, E., Andoh, A., Banu, R., Dorgbetor, W H K., Lubberding, 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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... 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
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