Fate of enteric viruses and bacteriophages in sequencing batch reactor treating domestic wastewater

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY - NGUYEN HOANG PHUONG THAO FATE OF ENTERIC VIRUSES AND BACTERIOPHAGES IN SEQUENCING BATCH REACTOR TREATING DOMESTIC WASTEWATER MASTER'S THESIS Hanoi, 2018 VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY  NGUYEN HOANG PHUONG THAO FATE OF ENTERIC VIRUSES AND BACTERIOPHAGES IN SEQUENCING BATCH REACTOR TREATING DOMESTIC WASTEWATER MAJOR: ENVIRONMENTAL ENGINEERING SUPERVISORS: Assoc Prof HIROYUKI KATAYAMA Prof HIROAKI FURUMAI Dr PHAM VAN QUAN Assoc Prof NGUYEN MANH KHAI Hanoi, 2018 ACKNOWLEDGEMENT Master thesis is completed at the Vietnam Japan University A completed study would not be done without any assistance Therefore, the author who conducted this thesis gratefully gives acknowledgment to their support and motivation during the time of doing this research I would first like to express my endless thanks and gratefulness to my supervisor Associate Professor Hiroyuki Katayama, Professor Hiroaki Furumai, Associate Professor Nguyen Manh Khai and Doctor Pham Van Quan His kindly support and continuous advices went through the process of completion of my thesis His encouragement and comments had significantly enriched and improved my work Without his motivation and instructions, the thesis would have been impossible to be done effectively I would like to offer my special thanks to Bac Ninh wastewater treatment plant, I have had the support and encouragement of them I would also like to express my greatest appreciation to Nagasaki Institute for supporting and facilitating the student's analysis of samples at the laboratory I would also like to thank the experts who were involved in the taking sample for this research: Duong Thu Thuy and Nguyen Tuan Khang Without their passionate participation and input, the thesis could not have been successfully conducted I wish to receive the contribution, criticism of the professors Sincerely thank i TABLE OF CONTENTS INTRODUCTION………………………………………………………………….1 Research purposes and significance Scope and objective CHAPTER LITERATURE REVIEW………………………………………….3 1.1 Review on enteric viruses 1.1.1.Sources of enteric viruses in the environment 1.1.2.Virus transport and survival in the environment 1.1.3.Waterborne enteric viruses and potential human diseases 1.1.4.The use of microbial indicator 1.2 Review on bacteriophages 10 1.2.1.The value of phages as surrogates for enteric viruses 11 1.2.2.F-specific RNA bacteriophages .12 1.2.3.Detected method for virus and phages .13 1.3 Review on wastewater treatment plant 18 1.3.1 State of wastewater treatment plant in the world and in Vietnam 18 1.3.2 Sequencing batch reactor treatment 19 CHAPTER MATERIALS AND METHODS……………………………… 23 2.1 Site description .23 2.2 Sample collection 24 2.3 Several indicators of analysis 26 ii 2.4 Sample concentration .28 2.5 Detection of E.coli and total coliforms by plate count .28 2.6 Detection of F-RNA phages by plaque assay 29 2.7 Method for quantitative genotyping of infectious FRNA phages coupled with MPN method 29 2.8 Recovery of viruses from sludge method 30 2.9 RT-PCR methods 31 2.10 Statistical analysis 34 CHAPTER RESULTS AND DISCUSSION………………………………….36 3.1 The occurrence of viruses and F-RNA phages 36 3.2 Reduction of viruses, F-RNA phages and microbial indicators 39 CHAPTER CONCLUSION………………………………………………… 45 REFERENCE ………………………………………………………………… 46 iii LIST OF TABLES Table 1.1 Advantage and disadvantage of culture and molecular methods .18 Table 2.1 The reaction mixture for RT 32 Table 2.2 The thermal condition of RT reaction 32 Table 2.3 The reaction mix for PCR 32 Table 2.4 The thermal condition for PCR 33 Table 3.1 Log10 mean concentration of F-RNA specific genotype 37 Table 3.2 Log10 concentration of E.coli and total coliforms 38 iv LIST OF FIGURES Figure 2.1 Influent tank .23 Figure 2.2 Reaction tank 23 Figure 2.3 Decanter 24 Figure 2.4 Effluent area 24 Figure 2.5 The cycle of Sequencing Batch Reactor Figure 2.6 Using a long bucket to take the sample 25 Figure 2.7 Using a syringe to take the sample 26 Figure 2.8 Thermal cycler 34 Figure 2.9 7500 fast Real-time PCR system 34 Figure 3.1 Concentration of E.coli 40 Figure 3.2 The concentration of total coliforms 40 Figure 3.3 The concentration of F-RNA phages genotype (a – GI, b-GII, c-GIII, dGIV) 41 Figure 3.4 Log 10 reductions of F-RNA phages genotype GI, GII, GIII, GIV and microbial indicators 43 v LIST OF ABBREVIATIONS CFU: Colony forming unit EPA: Environmental Protection Agency E coli: Escherichia coli GI: F-RNA Genotype I GII: F- RNA Genotype II GIII: F-RNA Genotype III GIV: F-RNA Genotype IV MPN: Most probable number NoVs: Norovirus PCR: Polymerase chain reaction PFU: Plaque forming units RT-PCR: Reverse transcriptase polymerase chain reaction RT-qPCR: Reverse transcriptase quantitative polymerase chain reaction WWTP: Wastewater treatment plant vi INTRODUCTION Research purposes and significance Viruses cause harm to humans because they are very small in size and therefore move in water and can cause disease with very low dose A group of viral pathogens, from within the human body, excrete through the feces, and overdisperse the drainage pipes Currently, researchers have identified several types of enteric viruses in the domestic effluent (Wong et al., 2012) mainly including enteroviruses (EVs), rotaviruses (RVs), adenoviruses (AdVs), noroviruses (NVs) hepatitis A virus (HAV) and astroviruses (AVs) The virus is responsible for some infectious diseases such as gastroenteritis, conjunctivitis, and respiratory disease, both developed and developing countries throughout the world Therefore, studying the behavioral characteristics, existence or inactivated of the viruses in the water treatment stages of the plant is the most important It is complicated and expensive to analyze all types of virus At present, many previous studies have tried to identify suitable viral indicators of wastewater treatment efficiency Several studies have proposed F- specific coliphages can be a good indicator monitoring the virus removal from wastewater (Tree et al., 2003; Duran et al., 2003), because there have similar morphological characteristics with many enteric viruses suggesting that they have the same single-stranded RNA, icosahedral shape, less than 50nm, and they are more persistent than bacteria FSpecific coliphages are bacteriophages that infect Escherichia coli cells These FRNA coliphages include MS-2 in group I, GA in group II, Qβ in group III and SP in group IV GII and GIII F-RNA phage genotypes was found primarily from human feces, while GI and GIV F-RNA phage genotype are involved in animal waste (Vinjé et al., 2004) The use of F- specific coliphages as indicators of the presence and behavior of enteric viruses and animal enteric viruses has always been attractive because of the easy of detection and low cost associated with plaque assay and for similarity to enteric viruses in terms of transport and survival characteristics Scope and objective This research studied the fate of enteric viruses and bacteriophages during the phase of sequencing batch reactor treatment plant Sequencing batch reactor (SBR) is a fill and draws activated sludge system In this system, each tank in the SBR system is filled during a discrete period of time and then operated as a batch reactor It means, once the reactor is full, it behaves like a conventional activated sludge system, but without a continuous influent or effluent flow The aeration and mixing are discontinued after the biological reactions are complete, the biomass settles, and the treated supernatant is removed The reason why this study chooses SBR system because the hydraulic retention time is time-based so there is no short-circuiting The influent and effluent can be pair so it is easy to observe the treatment efficiency using only a grab sample In addition, almost no research data in sequencing batch reactor for viruses’ removal So SBR system is the good target for an assessment of virus This study has three main objectives: To investigate the removal of enteric viruses and F-RNA phages in SBR system To evaluate the concentration of enteric viruses and F-RNA phages in activated sludge To find out the relationship between FRNA phage specific group I, II, III, IV and enteric viruses Influent 5/5 3.96 ± 0.41 5/5 4.59 ± 0.17 React 5/5 3.60 ± 0.22 5/5 4.46 ± 0.21 React 5/5 3.39 ± 0.33 5/5 4.40 ± 0.23 Effluent 5/5 2.55 ± 0.63 5/5 3.17 ± 0.58 Sludge 5/5 3.81 ± 0.26 5/5 4.64 ± 0.22 The concentration of E.coli and total coliforms was diferrent during the process of wastewater treatment plant In the influent sample, the E.coli and total coliforms' concentration was 12000 - 41000 CFU/ml, respectively It lower in reaction phase and the lowest was in the effluent sample The concentration of E.coli and total coliforms in the effluent sample were 670-2500 CFU/ml, respectively The concentration of E coli and total coliforms was significantly higher than that of FRNA phages 3.2 Reduction of viruses, F-RNA phages and microbial indicators - E.coli and total coliforms E.coli and total coliforms were detected in all samples with the concentration reduce from the influent through react 1, react 2, and lowest in effluent by 3.96 - 4.59; 3.60 - 4.46; 3.39 - 4.40; 2.55 - 3.17; 3.81 - 4.64 log10 CFU/ml, respectively (Fig 3.1 and Fig 3.2) 39 Figure 3.2 The concentration of total coliforms Figure 3.1 Concentration of E.coli E coli and total coliforms decreased through the treatment plant to show the efficiency of removal of microbial pathogens by SBR In the sludge sample, high levels of E coli and total coliforms showed that the microorganisms tend to adsorp into the sludge, or settle in the sludge - F-RNA coliphages genogroups To evaluate the differences in the behaviors of four different F-RNA coliphages genogroup during wastewater treatment, the log means concentration of each coliphage were calculated as shown in Fig 3.3 40 a, b, c, d, Figure 3.3 The concentration of F-RNA phages genotype (a – GI, b-GII, c-GIII, d-GIV) F-RNA phages genotype in wastewater reduced through the treatment stages also showed the efficiency of the wastewater treatment Mechanism of virus removal is adsorption to sludge or decomposition Virus tend to adsorp to sludge because of viruses have the surface charge, pH of the water and content of suspended solids in water effect to phages and viruses attached to particulate matter in water environments In this study, the results showed a change of phage’s concentration in sludge, and variations in among phages F-RNA phages genotype From graphs above, the concentration was changed in water and in sludge were observed through the phase of the SBR It is possible to divide the varying concentrations follow the phase of the tank into three phases Section one from S1 –R1 corresponds to the filling 41 operation Section two from R1 –R2 corresponds to reaction phase Section three from R2 –S2 corresponds to settling and draw phases It can be seen that for GI F-RNA phage, the comparison between the line concentration of liquid and solid is reversed in all stages (Fig 3.3 a) On stage one, the GI F-RNA phage's concentration in the liquid phase reduces while increase in solid phase, the result implies that the phages adsorp from water to sludge On the second and the third stage, it is opposite of stage one, it performed the desorp of phages from sludge to water For GII F-RNA (Fig 3.3 b), in general, the concentration of phages in the water phase decreases while the concentration in the sludge increases during the reaction stage In detail, there is decomposition in stage one based on the trend of phages' concentration decrease on both liquid and solid phase On the second stage showed that the phages adsorp from water to the sludge occurs On the last stage, which shows that the phages decompose in water because the concentration of phages in sludge is relatively stable, but the concentration of phages in the liquid is still reduced As can be seen from the graph of GIII F-RNA phage (Fig 3.3 c), the concentrate of phages in water was decline and similary with GIII F-RNA phage On stage one, the phages' concentration was decreased in both phase, it performs that the decomposition of virus occurs On the second and third stage, base on the graph shows the adsorption and desorption base on the reverse between phages' concentration in liquid and solid phase, respectively For GIV F-RNA phage (Fig 3.3 d), the concentration of phages in the sludge was very low and stable, the concentration of virus in the water gradually decline until finish reaction phase and then grow up in effluent phase The graph showed that on stage one and stage two occurred the decomposition Overall, in four group of F-RNA phages, GI F-RNA phage showed the adsorption but did not the decomposition GII F-RNA phage and GIII F-RNA phage showed 42 both mechanisms of virus removal While GIV F-RNA phage just shows the decomposition Base on the concentration in influent and effluent sample, the reduction ratio of FRNA phages and microbial indicator were determining and showing on Figure 3.4 Figure 3.4 Log 10 reductions of F-RNA phages genotype GI, GII, GIII, GIV and microbial indicators Log10 reduction of GI show the lowest value of 0.14 ± 0.11 (n=4) (ANOVA, P>0.05); and the highest value is GIII 1.24 ± 0.097 (ANOVA, P0.05); 0.96 ± 0.70 log10 (ANOVA, P