HUMAN ADENOVIRUS REMOVAL IN WASTEWATER TREATMENT AND MEMBRANE PROCESS By Ziqiang Yin A DISSERTATION Submitted to Michigan State University i n partial fulfillment of the requirements for the degree of Environmental Engineering Doctor of Philos ophy 2015 ABSTRACT HUMAN ADENOVIRUS REMOVAL IN WASTEWATER TREATMENT AND MEMBRANE PROCESS By Ziqiang Yin Occurrence of human pathogenic viruses in environmental waters (i.e., surface waters, groundwater, drinking water, recreational water, and wastew ater) raises concerns regarding the possibility of human exposure and waterborne infections. Presence of virus in water and wastewater is a difficult problem for environmental engineers because of prevalence, infectivity, and resistance of viruses to disin fection. On the other hand, it has been suggested that development of membrane Technology in treating municipal wastewater, such as membrane bioreactors, provide s high quality effluents appropriate for water reuse. Removal of human adenovirus 40 (HAdV 40) by hollow fiber ultrafiltration (UF, membranes was elvauted in humic acid (model dissolved species), SiO 2 microspheres (model suspended species) and a mix of these constituents. Three separate effects are identified: 1) increased removal due to pore blockage by dissolved species; 2) decreased removal due to cake - enhanced accumulation of viruses near membrane surface; and 3) increased removal by the compo site cake acting as a secondary membrane. Comp aring to the extent of fouling, feed water composition and membrane pore size together plays more important role in virus removal. Pore blockage improves virus removal while cake formation can either increase o r decrease virus removal depending on the relative permeability of the cake. Pressure relaxation and permeate backwash are two commonly used physical methods for membrane fouling mitigation in membrane bioreactor (MBR) systems. In order to assess the impac t of these methods on virus removal by MBRs, experiments were conducted in a bench - scale submerged MBR treating synthetic wastewater. The membranes employed were hollow fibers with the nominal pore size of 0.45 µm. The experimental variables included durat ions of the filtration ( ), pressure relaxation ( ) and backwash ( ) steps. Both pressure relaxation and permeate backwash led to significant reductions in virus removal. For the same value of , longer filtr ation/relaxation cycles (i.e. larger ) led to higher transmembrane pressure ( ) but did not have a significant impact on virus removal. A shorter backwash ( = 10 min) at a higher flow rate ( = 40 mL/min) re sulted in more substantial decreases in and virus removal than a longer backwash ( = 20 min) at a lower flow rate ( = 20 mL/min) even though the backwash volume ( ) was the same. Virus removal returned to pre - cleaning levels within 16 h after backwash was applied. Moderate to strong correlations ( = 0.63 to 0.94) were found between and virus removal. Virus adsorption to sludge particles has been suggested as one of the major mech anisms of virus removal. Our results showed that adsorption of HAdV to primary and secondary sludge conformed to Freundlich isotherm, and it exhibited very similar behavior in the two types of sludge. More HAdV was desorbed from primary sludge during seque ntial desorption experiments, but the difference was not statistically significant. Greater HAdV adsorption was observed when sludge filtrate was used as solute compared to DI water. iv ACKNOWLEDGEMENTS I first would like to express my sincerest gratitude to my parents f or their emotional and financial support during the seven years I spent at Michigan State University. I would like to acknowledge my dissertation committee members: Dr. Xagoraraki, Dr. Tarabara, Dr. Voice and Dr. Bruening , for their guidance , support, and encouragement while I was pursuing my Ph.D. This work would not be accomplished without them. I would like to thank Ms. Lori Hasse and Ms. Margaret Conner for their dedicative administrative support. I would like to thank Yang - lyang Pan and Joseph Nguyen for their technical and lab support. I also would like to thank my colleagues and frien ds: Mariya Munir, Hang Shi, Bin Guo, and Amira Oun for countless productive discussion s and generous help. v TABLE OF CONTENT S LIST OF TABLES ................................ ................................ ................................ ................. viii LIST OF FIGURES ................................ ................................ ................................ .................. ix K EY TO ABBREVIATIONS ................................ ................................ ................................ .... xi CHAPTER 1 ................................ ................................ ................................ .............................. 1 BACKGROUND ................................ ................................ ................................ ....................... 1 1.1. Viruses of Concern in the U.S . ................................ ................................ ............................ 1 1.1.1. Waterborne v iruses and p otential h uman d iseases ................................ .................... 1 1.1.2. Waterborne o utbreaks r elated to v iruses ................................ ................................ ... 3 1.2. Source and Fates of Viruses in the Environment ................................ ................................ 5 1.2.1. Sources of v iruses in the e nvironment ................................ ................................ ...... 5 1.2.2. Viru ses as m icrobial s ource t racking t ools ................................ ................................ 6 1.2.3. Viruses in n atural w ater b odies, s ediments, and s oils ................................ ............... 7 1.2.4. Virus s urvival in the e nvironment ................................ ................................ ............. 8 1.2.5. Virus t ransport in the e nvironment ................................ ................................ ........... 9 1.3. Detection Metnods ................................ ................................ ................................ ............ 11 1.4. Fate of Viruses during Water Treatment ................................ ................................ ........... 14 1.4.1. Fate of v iruses during f ull - s cale w ater t reatment ................................ .................... 14 1.4.2. Virus i nactivation ................................ ................................ ................................ .... 15 1.5. Fate of Viruses in Wastewater Treatment Systems ................................ ........................... 16 1.5.1. Virus removal in f ull - s cale w astewater u tilities ................................ ...................... 16 1.5.2. Virus r emovals in b ench and p ilot - s cale MBR s ystems ................................ .......... 18 1.5.3. Viruses in b iosolids ................................ ................................ ................................ . 20 1.5.4. Bacterial v iruses ( p hages) in w astewater ................................ ................................ 20 1.6. Viral Risk Assessment ................................ ................................ ................................ ....... 22 1.7. Summary and Conclusions ................................ ................................ ............................... 24 APPENDIX . . . 2 6 R E FERENCES ... ...................................................................... ............................... 40 CHAPTER 2 ................................ ................................ ................................ ............................ 6 3 LITERATURE REVIEW: MEMBRANE BIOREA C TORS FOR WATER REUSE IN THE UNITED STATES ................................ ................................ ................................ .................... 6 3 Abstract ................................ ................................ ................................ ................................ .... 6 3 2.1. Water Reuse in the United States ................................ ................................ ...................... 6 4 2.2. Water Reuse Standards ................................ ................................ ................................ ...... 6 6 2.3. Membrane Bioreactors Technology for Water Reuse ................................ ....................... 6 7 2.4. Membrane Fouling: Major Challenge of MBR Application ................................ ............. 6 8 2.5. Pollutant Removal in MBR Systems ................................ ................................ ................ 7 1 2.5.1. Removal of p hysical and c hemical p ollutants in MBRs ................................ ......... 7 2 2.5.2. Removal of p athogens in MBRs ................................ ................................ ............. 7 3 2.6. Comparison between Conventional Activated Sludge (CAS) system and MBR ............. 7 5 2.7. Applications of MBR with Water Reuse in the U.S. ................................ ......................... 76 2.8. Conclusions ................................ ................................ ................................ ....................... 77 APPENDIX 79 REFERENCES ................................ ................................ ................................ ........................ 86 vi CHAPTER 3 ................................ ................................ ................................ ............................ 9 6 HUMAN ADENOVIRUS REMOVAL BY HOLLOW FIBER MEMBRANES: EFFECT OF MEMBRANE FOULING BY SUSPENDED AND DISSOLVED MATTER ........................ 9 6 Abstract ................................ ................................ ................................ ................................ .... 9 6 3.1. Introduction ................................ ................................ ................................ ....................... 9 8 3.2. Materials and Methods ................................ ................................ ................................ .... 10 1 3.2.1. Cell culture experiment and virus incubation ................................ ....................... 10 1 3.2.2. Membrane preparation ................................ ................................ .......................... 10 1 3.2.3. Foulant preparation and particle size ................................ ................................ .... 10 2 3.2.4. Membrane filtration experiment ................................ ................................ ........... 10 2 3.2.5. DNA extraction and quantitative polymerase chain reaction (qPCR) .................. 10 3 3.2.6. Inhibition of qPCR by humic acid ................................ ................................ ........ 10 4 3.2.7. Scanning electron microscopy (SEM) imaging of membranes ............................ 10 4 3.2.8. Membrane challenge tests ................................ ................................ ..................... 104 3.3. Results and Discussion ................................ ................................ ................................ ... 105 3.3.1. Characterization of membranes and model foulants ................................ ............. 105 3.3.2. Inhibition of qPCR by humic acid ................................ ................................ ........ 106 3.3.3. Membrane fouling and transmembrane pressure buildup ................................ ..... 106 3.3.4. Removal of human adenovirus 40 by clean and fouled membranes .................... 108 3.4. Conclusions ................................ ................................ ................................ ..................... 11 1 APPENDIX 11 3 REFERENCES ................................ ................................ ................................ ...................... 125 CHAPTER 4 ................................ ................................ ................................ .......................... 131 EFFECT OF PRESSURE RELAXATION AND MEMBRANE BACKWASH ON VIRUS REMOVAL IN A MEMBRANE BIOREACTOR ................................ ................................ . 131 Abstract ................................ ................................ ................................ ................................ .. 131 4.1. Introduction ................................ ................................ ................................ ..................... 132 4.2. Materials and Methods ................................ ................................ ................................ .... 135 4.2.1. Cell culture experiment and virus incubation ................................ ....................... 135 4.2.2. Membrane preparation ................................ ................................ .......................... 135 4.2.3. Bench - scale submerged MBR ................................ ................................ ............... 136 4.2.4. Fouling and backwash experiments ................................ ................................ ...... 136 4.2.5. DNA extraction and quantit ative polymerase chain reaction (qPCR) .................. 137 4.2.6. Inhibition test of qPCR ................................ ................................ ......................... 138 4.3. Results and Discussion ................................ ................................ ................................ ... 138 4.3.1. Membrane fouling and transmembrane pressure buildup ................................ ..... 138 4.3.2. Virus removal ................................ ................................ ................................ ........ 140 4.3.3. Relationship between transmembrane pressure and virus removal ...................... 143 4.4. Conclusions ................................ ................................ ................................ ..................... 143 APPENDIX 14 5 REFERENCES ................................ ................................ ................................ ...................... 157 CHAPTER 5 ................................ ................................ ................................ .......................... 163 ADSORPTION AND DESORPTION OF HUMAN ADENOVIRUS TO PRIMARY AND SECONDARY SLUDGE ................................ ................................ ................................ ....... 163 Abstract ................................ ................................ ................................ ................................ .. 163 5.1. Introduction ................................ ................................ ................................ ..................... 164 5.1.1. Viruses in the wa stewater ................................ ................................ ...................... 164 5.1.2. Virus sorption mechanisms ................................ ................................ ................... 164 5.1.3. Virus sorption in activated sludge and biosolids ................................ ................... 168 vii 5.2. Material and Methods ................................ ................................ ................................ ..... 1 69 5.2.1. Human adenovirus preparation ................................ ................................ ............. 1 69 5.2.2. Sludge sampling and processing ................................ ................................ ........... 17 0 5.2.3. DNA extraction and qPCR assay ................................ ................................ .......... 170 5.2.4. Equilibrium time determination ................................ ................................ ............ 170 5.2.5. Optimal solid/liquid ratio determi nation ................................ ............................... 171 5.2.6. HAdV adsorption ................................ ................................ ................................ .. 171 5.2.7. Sorption isotherm experiments ................................ ................................ ............. 17 2 5.2.8. Sequenti al desorption experiments ................................ ................................ ....... 17 2 5.3. Results and Discussion ................................ ................................ ................................ ... 17 3 5.3.1. Equilibrium time and optimal S/L ratio ................................ ................................ 17 3 5.3.2. HAdV adsorption to sludge using DI water and sludge filtrate as solute ............. 17 4 5.3.3. Adsorption isotherm of HAdV ................................ ................................ .............. 17 5 5.3.4. HAdV desorption from sludge particles ................................ ............................... 17 6 5.4. Implications ................................ ................................ ................................ ..................... 177 APPENDIX ... 1 79 REFERENCES ................................ ................................ ................................ ...................... 18 7 viii LIST OF TABLES Table 1 - 1. Human Viruses in the Environmen tal Protection Agency Contaminant Candidate Lists (CCL) ................................ ................................ ................................ .............................. 2 7 Table 1 - 2. Summary of Virus Surface Properties Affecting Sorptive Removal from Water ... 2 8 Table 1 - 3. Virus Removal in Full - Scale Membrane Bioreactors ................................ ............ 29 Table 1 - 4. Virus removal in bench and pilot - scale membrane bioreactors ............................. 3 0 Table 1 - 5. Virus occurrence in dewatered sludge and class B biosolids ................................ . 3 1 Table 1 - 6. Dose response models for enteric viruses ................................ .............................. 3 2 Table 2 - 1. Water quality criteria of EPA guideline for water reuse (EPA 2012) ..................... 8 0 Table 2 - 2. Pollutant removal i n selected full - scale MBR plants in the U.S. ........................... 8 1 Table 2 - 3. Bacteria removal in full - scale MBRs ................................ ................................ ..... 8 2 Table 2 - 4. Virus removal in full - scale MBRs ................................ ................................ .......... 8 3 Table 2 - 5. Selected MBR wastewater treatment faciliti es in the U.S. with water reuse ......... 8 4 Table 3 - 1. Characteristics of hollow fiber membranes ................................ .......................... 1 1 4 Table 3 - 2. Sampling protocols in fouling experi ments with different membranes and feed waters of different compositions ................................ ................................ ............................ 1 1 5 Table 3 - 3. Log removal of probe particles in challenge tests with the UF, MF1, and MF2 membranes ................................ ................................ ................................ ............................. 116 Table 4 - 1. Effect of membrane fouling mitigation method s on virus removal in submerged MBRs .......................... .......................................................................................................... . 1 4 6 Table 4 - 2. Composition of the syntheti c wastewater ................... ............................... .. . ..... . .. 1 4 7 Table 4 - 3. Parameters of pressure relaxation and backwash ..................... .................... .... . ... 1 4 8 Table 5 - 1. Virus partitioning/removal due to sorption in activated sludge ............................ 18 0 Table 5 - 2. Virus desorption from sludge/biosolids ................................. ............................... 18 1 Table 5 - 3. TSS and DOC in primary and secondary sludg .............................................. ............. 18 2 Table 5 - 4. Virus concentration in supernatant with different S/L ratio ................................ . 18 3 Table 5 - 5. Comparison of DI water and sludge filtrate as solute for HAdV adsorption ... 18 4 ix LIST OF FIGURES Figure 1 - 1. Sources of viruses in the environment ................................ ................................ . 33 Figure 1 - 2. Summary of virus elution and detection methods ................................ ................ 34 Figure 1 - 3. Adenovirus removal in full - scale wastewater treatment plants ............................ 35 Figure 1 - 4. Enterovirus removal in full - scale waste water treatment pl ants .......................... 3 6 Figure 1 - 5. Norovirus I removal in full - scale waste water treatment plants ........................... 3 7 Figure 1 - 6. Norovirus II removal in full - scale waste water treatment plants ......................... 3 8 Figure 1 - 7. Virus removal as a function of membrane pore size in bench and pilot scale MBR systems ................................ ................................ ................................ ................................ ..... 39 Figure 3 - 1. Schematic of the experimental apparatus ................................ ........................... 1 1 7 Figure 3 - 2. Pa rticle size distribution of model foulants ................................ ........................ 1 1 8 Figure 3 - 3. SEM micrographs of cross - sections (A - C) and the planar view of the separation layer (D F) of the three membranes ................................ ................................ .................... 1 19 Figure 3 - 4. Transmembrane pressure as a function of time during filtration of HAdV 40 suspension ( - - , - - , - - ) and HAdV - seeded feeds containing SiO 2 microspheres ( - - ), humic acid ( - - ), and SiO2/HA mixture ( - - ) by three membranes of different nominal pore si zes: a) 0.04 µm, b) 0.22 µm, and c) 0.45 µm. ................................ ................................ ................... 1 2 0 Figure 3 - 5. Blocking laws applied to filtration of SiO 2 microspheres and humic acid by UF and MF2 membranes ................................ ................................ ................................ .............. 1 2 1 Figure 3 - 6. Removal of HA dV 40 from DI water by three membranes of different nominal pore sizes ................................ ................................ ................................ ................................ 1 2 2 Figure 3 - 7. Comparison of HAdV 40 removal from DI water, suspension of SiO 2 microspheres, solution of humic acid, and SiO 2 /HA mixture by three mem branes of different nominal pore sizes : a) 0.04 µm, b) 0.22 µm, and c) 0.45 µm. . ................................ .............. 1 2 3 Figure 3 - 8. Schematic illustration of effects of fouling on HAdV 40 removal by ultrafiltration (A, B, C) and microfiltration (D, E, F) m embranes under conditions of fouling by dissolved species (A, D), suspended particles (B, E) and by both of the se foulants (C, F) ................... 1 2 4 Figure 4 - 1. Schematic of the submerged MBR ................................ ................................ ..... 1 49 Figur e 4 - 2. Transmembrane pressure as a function of filtration time and the effect of backwash ................................ ................................ ................................ ................................ 1 5 0 Figure 4 - 3. Effects of pressure relaxation and backwash on the removal of HAdV 40 in submerged MBR operated under three different filtration / pressure re laxati on schedules and x backwash protocols : : A: exp. 1; B: exp. 2; C: exp. 3 ............................................. ...... ......... 1 5 1 Figure 4 - 4. Decrease in virus removal as a result of backwash for two different backwash formats ....... .................. ...........................................................................................................1 5 3 Figure 4 - 5. Decrease in virus removal as a result of pressure relaxation for two different formats of the filtration/relaxation (F/R) cycle..... ........................................... ... ...................1 5 4 Figure 4 - 6. Correlations between virus removal and transmembrane pressure in experiments....... ........................ ................................................................... ..........................1 5 5 Figure 5 - 1. Adorption isotherm curves (1) primary sludg e; (2) secondary sludge ............... 18 5 Figure 5 - 2. Percentage of HAdV desorbed from sludge part icles in sequentia l experiments : (1) Primary sludge; (2) Secondary sludge ................................ ................................ ............. 18 6 xi KEY TO ABBRE VIATIONS BOD - Biochemical Oxygen Demand CAS - Conventional Activated Sludge COD - Chemical Oxygen Demand EBPR - Enhanced Biological Phosphorus Removal EPS - Extracellular Polymeric Substances FAO - Food and Agriculture Organization GE - General Electric IWMI - International Water Management Institute HA - Humic Acid HAdV - Human Adenovirus HRT - Hydraulic Retention Time MBR - Membrane Bioreactor MLSS - Mixed Liquor Suspend ed Solids NRC - National Research Council NTU - Nephelometric Turbidity Unit PVDF - Polyvinylidene fluoride SEM - Scanning Electron Microscope SMP - Soluble Microbial Products SNdN - Simultaneous Nitrification and Denitrification SRT - Solids Retention Tim e TMP - Trans - membrane Pressure TOC - Total Organic Carbon TKN - Total Kjeldahl Nitrogen TN - Total Nitrogen xii TP - Total Phosphate TSS - Total Suspended Solids USEPA - United States Environmental Protection Agency UV - Ultraviolet WHO - World Health Organ ization WWTP - Wastewater Treatment Plant 1 CHAPTER 1 BACKGROUND 1. 1. Viruses of Concern in the U.S. 1. 1.1. Waterborne v iruses and p otential h uman d iseases Viruses are the most abundant microorganisms on t he earth (Madigan and Martinko 2006). It has been suggested that more than 150 types of enteric viruses are excreted in human feces and may be present in c ontaminated waters (Wong et al. 2012a; Leclerc et al. 2000; Havelaar et al. 1993). Enteric viruses are usually transmitted to humans by oral ingestion (Tanni et al. 1992). Infection by viruses may lead to various diseases, including gastroenteritis, heart anomalies, meningitis, conjunctivitis, hepatitis, and respiratory diseases (Crites and Tchobanoglous 1998; Swenson et al. 2003). Waterborne viral infec tions can be fatal to sensitive populations such as children, the elderly, and the immune - compromised. Waterborne disease statistics reflect a growing global burden of infectious diseases from contaminated drinking water, while ingestion of surface water d uring recreational activities is also a common exposure pathway to viruses and other pathogens. Viruses are contaminants of concern that may be regulated in the future, as indicated by their presence on Environmental andidate lists (Table 1 - 1). Table 1 - 1 also includes the classification for these waterborne viruses. Generally, there are two major systems for virus classification. One system is authorized and organized by the International Committee on Taxonomy of Virus es (ICTV). Based on both genome type and sequence similarity, ICTV classification divides viruses in orders ( - virales), families ( - viridae), subfamilies ( - virinae), genera ( - virus), and species (Korsman et al. 2012). The current (2012) ICTV taxonomy includ es 7 orders, 96 families, 22 subfamilies, 420 genera, and 2618 species (ICTV, 2012). Another system is called Baltimore classification, which 2 classifies viruses into seven groups with different types of hosts (animal, plant, bacteria, algae, fungi and prot ozoa) on the basis of genome type and replication strategy. Most virus families are included in Groups I V, whereas only a few families belong to Groups VI and VII (Dimmock et al. 2001). Human adenoviruses are important opportunistic pathogens in immuno compromised patients (Wadell 1984) and have been identified as etiological agents in several waterborne outbreaks (Foy et al . et al. 1979; Martone et al. 1980; Kukkula et al. 1997; Papapetropoulou and Vantarakis 1998; Borchardt et al. 2003a) . Diseases caused by human adenoviruses include conjunctivitis, ocular infections, gastroenteritis, respiratory disease, encephalitis, pneumonia, genitourinary infections, and pharyngoconjunctival fever. The potential health risk to infants, children and a dults, associated with adenovirus waterborne transmission are confirmed by the scientific community ( Irving and Smith 1981; Albert 1986; Uhnoo et al. 1986; Adrian et al. 1987; Hurst et al. 1988; Krajden et al. 1990; Cruz et al. 1990; Enriquez et al. 1995; Horwitz 1996; Foy 1997; Bon et al. 1999; Borchardt et al . 2003a; Swenson et al. 2003). It has been reported that enteroviruses are responsible for most outbreaks of enteroviral meningitis (Abzug et al. 2003; Rotbart 2000). Poliovirus is a type of human en terovirus mainly causing poliomyelitis (Madaeni et al. 1995). Coxsackievirus usually causes - foot - and - immune systems. Echovirus is a subspecies of enterovirus B, and it is a us ual cause of aseptic meningitis ( Martinez et al. 2012; Xiao et al. 2013 ). Symptoms of infection by hepatitis A virus vary greatly, and severe cases of infection can cause death. Person - to - person contact is an important transmission path in addition to feca lly contaminated food and water ( Morace et al. 2002; Cuthbert 2001 ). Hepatitis virus has a prolonged incubation period in cell cultures and polymerase chain reaction ( PCR) is suggested as a preferable method for HAV detection (Divizia et al. 1998). 3 Calici viruses cause various diseases in animals, including gastroenteritis, respiratory infections, vesicular lesions, hemorrhagic disease, while the associated disease in humans is mainly gastroenteritis (Farkas et al. 2008). Noroviruses are the most common eti ologic agents in caliciviridae family. They are highly contagious, and the required dose for viral infection is very low (Ausar et al. 2006). One challenge in norovirus studies is that high concentrations of noroviruses cannot be easily produced since they are not culturable (Farkas et al. 2008). Rotavirus has been recognized as one of the most common causes of acute infec tious gastroenteritis (Marshall 2009) and the leading cause of severe, dehydr ating diarrhea in children (WHO 2007). Outbreaks of viral ga stroenteritis caused by rotaviruses have been reported in both i nfants and adults (Craun et al. 2010; Anderson and Weber 2004; Siqueira et al. 2010), and rotaviruses might be responsible for more than 50% of enteritis among infa nts worldwide (Fenner and Wh ite 1976). 1. 1.2. Waterborne o utbreaks r elated to v iruses It has been reported that 1.5 - 12 million people die per year from waterborne diseases (Gleick 2002 ; WHO 2004). Most of the waterborne outbreaks in the US have been related to microbial agents (Moo re et al . 1993; Kramer et al . 1996; Levy et al . 1998; Barwick et al . 2000; Lee et al . 2002; Yoder et al . 2004; Liang et al . 2006), and over the last decade, thousands of people in the United States have experienced waterborne diseases. The majority of the outbreaks involved unidentified agents. The Environmental Protection Agency suspects that many of the outbreaks due to unidentified sources were cause d by enteric viruses (USEPA 2006). Ground water is an important transmission route for wat erborne viral i nfections (USEPA 2006). The majority of outbreaks associated with drinking water are caused by water from wells, while outbreaks associated with recreational water mainly occur in natural water bodies. Since 1980, over 70 outbreaks of diseases in the Unite d States 4 reported by the CDC have been attributed to viruses, and it is estimated that the actual number of outbreaks is a lot higher. It is believed that the role of viruses associated with waterborne disease is underestimated since their occurrences are under - reported and it is difficult to specify the agents (Mena et al. 2007). Noroviruses (Norwalk - like virus) appear to be the most common aetiological agents of gastroenteritis in the United States and are responsible for more than half of both recreatio nal and drinking water outbreaks ( Blackburn et al. 2004; Yoder et al. 2008a; Brunkard et al. 2011; Barwick et al. 2000; Lee et al. 2002; Yoder et al. 2004; Dziuban et al. 2006; Yoder et al. 2008b; Hlavsa et al. 2011 ). Outbreaks caused by Hepatitis A viruse s are also frequently reported by CDC and are mostly associated with drinking water as opposed to recreational water exposure ( Kramer et a l. 1996; Moore et al. 1993; Yoder et al. 2008a; Brunkard et al. 2011; Mahoney et al . 1992 ). Three outbreaks reported b y CDC were caused by adenoviruses. One was in 1982 and two were in 1991. All were related to recreational water, and the associated diseases include conjunctivitis and Pharyngitis (Turner et al. 1987; Moore et al. 1993) . Enteroviruses ( coxsackievirus, echo virus ) were reported as aetiological agents in three outbreaks (Hejkal et al. 1982; Levine et al. 1990; Dziuban et al. 2006), two of which were related to recreational water. Associated diseases include meningitis and gastroenteritis . Rotaviruses were the cause of one outbreak in Colorado , and tap water was identified as the contamination source ( Hopkins et al. 1985 ). Outbreaks of hepatitis E were reported in other countries (Corwin et al. 1996), but the United States is considered a non - endemic area for he patitis E (Favorov et al. 1992; Favorov et al. 1999; Aggarwal and Krawczynski 2000), and et al. 2010). However, sporadic cases of hepatitis E infection have been observed (Tsang et al . 2000; Kwo e t al. 1997; Munoz et al. 1992), and some of the patients had no history of travelling outside the U.S. (Tsang et al. 2000). Swine are known as a reservoir of hepatitis E, and also a potential source for virus trans mission to human (Colson et al. 2010; Dong et al. 2011). 5 1. 2. Source and Fates of Viruses in the Environment 1. 2.1. Sources of v iruses in the e nvironment The sources and reservoirs of human viruses are shown in Figure 1 - 1. Human enteric viruses are frequently found in surface water, and the sourc es of viruses could be effluent from wastewater treatment plants, combined sewer overflows, leaching septic systems, and runoff from agriculture areas. Runoff and infiltration during precipitation events can lead to viral contamination of surface and groun dwater. In the case of permeable soils, the most likely route of pollutant transfer is through the soil to groundwater. Preferential flow paths caused by plant roots, cracks, fissures and other natural phenomena can rapidly move viral contaminants to shall ow groundwater. Wastewater is one of the most concentrated sources of infectious viruses (Puig et al . 1994, Castignolles et al . 1998). The estimated mean concentration of enteric viruses in wastewater in the United States is approximately 7000 infectious v iruses per liter (Melnick et al. 1978), and the highest concentrations of viral particles can reach 10 9 per liter (da Silva et al. 2007; Kuo et al. 2010; Simmons et al. 2011). Wastewater utilities may release viruses to environmental waters via treated eff luent discharge and biosolids that are land applied. During rainfall events, untreated sewage and wastewater may be directly discharged into surface water in combined sewer overflows (Donovan et al. 2007). Fecal contamination from livestock manure handlin g and storage facilities is one of the most important sources of groundwater m icrobiological pollution (USEPA 2006). Manure and other animal wastes contain high concentrations of infectious zoonotic viruses, protozoa, and bacteria (Meslin 1997; Slifko et a l. 2000; Sobsey et al. 2001; Hubalek 2003; Gannon et al. 2004; Cliver and Moe 2004; Palmer et al. 2005). Zoonotic viruses from animals may cause diseases in humans. For example, hepatitis E is considered as a zoonotic virus, of which the potential transmis sion from animal, such as swine, to human has been proposed ( Clayson et al. 1996; Wu et al. 2000 ). 6 1. 2.2. Viruses as m icrobial s ource t racking t ools Traditional microbial indicators are widespread in the environment, and the related measurements are simpl e. However, the most significant deficiency of E.coli and enterococci as MST tools is lack of host specificity (Ahmed et al. 2007; Gordon et al. 2001). Microbial source tracking (MST) is a relatively new, fast developing technology that allows people to di scriminate among possible sources of fecal contamination in the environment (Hagedorn et al. 2011). A number of microorganisms have been proposed as candidate tools for MST. Human adenovirus (HAdV), human enterovirus (HEV), and human polyomavirus (HPyV), h ave been suggested as potential MST tools indicating human pollution sources (Harwood et al. 2009; Noble et al. 2003; Ahmed et al. 2010). Fong et al. (2005) characterized HAdVs and HEVs as sound library - independent indicators that can be used for the ident ification of water pollution sources. After analyzing pig slaughterhouse slurries, urban sewage and river water samples, Hundesa et al. (2006 and 2009) suggested that porcine adenoviruses (PAdVs) detection provides a valuable MST approach. Also, HPyVs are highly human specific, so that their detection provides a reliable indication of contamination from a human source (Harwood et al. 2009). Bovine adenovirus (BAdV) and bovine enterovirus (BEV) were proposed for use in identifying agricultural water pollutio n sources (Ahmed et al. 2010; Fong et al. 2005). Bovine polyomavirus (BPyV) has been characterized as a particularly robust MST tool (Hundesa et al. 2010) that might perform better than BAdV at sites where manure is a suspected source of con tamination (Won g and Xagoraraki 2011). Moreover, some types of bacteriophages, such as F RNA specific phage (Lee et al. 2009; Smith et al. 2006; Stewart et al. 2 006; Gourmelon et al. 2010), were also suggested as potential MST tools. The occurrence and concentration of h uman and animal viruses are fairly low in fresh water bodies. In order to make viruses detectable and efficiently use them as MST tools, a concentration procedure is usually required involving filtration of large amounts of water during sampling. 7 1. 2.3. V iruses in n atural w ater b odies, s ediments, and s oils Numerous studies have found human enteric viruses in surface water in many countries including well developed, industrialized countries (De Paula et al . 2007; Xagoraraki et al . 2007; Jiang et al . 2007; M iagostovich et al . 2008; Chen et al . 2008; Shieh et al . 2008; Costan - Longares et al . 2008). As an example, occurrences of enteric viruses have been reported in fresh water in the Great Lakes region. Human adenoviruses were the most frequently detected viru ses at Great Lakes beaches ( Fong et al. 2007; Aslan et al. 2011; Wong et al. 2009a; Xagoraraki et al. 2007 ). Enteroviruses and rotaviruses have also been detected at some beaches, but tw o studies involving noroviruses failed to detect them. Viruses are al so found in sediments. When microorganisms enter the natural water, some of them adsorb on the surface of particles that can settle or re - suspend into the water column, since adsorption may be reversible. Re - suspension of enteric viruses in waters impacted by fecal contamination could pose a potential risk to human health (De Flora et al. 1975). Ferguson et al. (1996) suspected that sediments can act as reservoirs for enteric viruses. They took samples from an urban estuary and detected viruses primarily in water and top sediment, whereas no viruses were found in the bottom sed iment. Human enteric viruses have been found in ground water (Abbaszadegan et al . 2003; Fout et al . 2003; Borchardt et al. 2003b; Lieberman et al . 1995; Davis and Witt 1998). In a nati onwide study, samples from 448 groundwater sites in 35 states were analyzed for enteroviruses, rotaviruses, hepatitis A viruses and noroviruses. Viral nucleic acid was present in 31% of samples (Abbaszadegan et al. 2003). Human enteric viruses (enterovirus es, hepatitis A viruses, Norwalk viruses, reoviruses or rotaviruses) were detected in 16% of 29 groundwater sites sampled over one year (Fout et al. 2003). Borchardt et al. (2003b) tested 50 private household wells in Wisconsin four times per year and foun d that four wells (8%) were positive for hepatitis A viruses or rotaviruses, noroviruses and enteroviruses. In an earlier study (Lieberman et al. 1995) in which 30 public water supply wells were examined, the 8 authors reported that 24% of the samples were p ositive for culturable viruses. Also, the US Geological Survey (Davis and Witt 1998) reported about 8% of wells positive for culturable human viruses. Viruses and other microorganisms can survive for several months in soil and ground water when temperatu res are low and soils are moist (Yates et al. 1985; Jansons et al. 1989; Straub et al. 1993; Robertson and Edberg 1997), increasing risk due to groundwater contamination. Presumably, most microbial transport occurs in saturated soil (Jamieson et al. 2002; Powelson and Mills 1998) or by preferential flow (Shipitalo and Gibbs 2000; Mawdsley et al. 1995). Penetration of viruses to depths as great as 67 m (220 ft) and horizontal migration as far as 408 m (1,339 ft) in glacial till and 1,600 m (5,240 ft) in frac tured limestone have b een reported (Keswick and Gerba 1980; Robertson and Edberg 1997). 1. 2.4. Virus s urvival in the e nvironment Type of soil, particle size distribution, clay composition, soil organic content, presence of dissolved or colloidal organic carbon, solution chemistry, metal oxides, degree of saturation of the solid media, ionic strength, temperature, pH, light, presence of air - water interfaces, and biological factors are primary factors influencing virus survival and trans port in the environ ment (Gerba 2007; Gerba et al. 1975; Gerba and Bitton 1984; Sobsey et al. 1986; Yates and Yates 1988; Gerba and Rose 1990; Schijven and Hassanizadeh 2000; Jin and Flury 2002; Zhuang and Jin 2003). In water, virus survival mainly depends on temperature, exp osure to UV and presence of microbiological flora (B osch 2006). In seawater at 15 ° C , polio and adenovirus 40 and 41 can survive for many days. Reduction of 3 logs, 1.4 and 1.6 logs, respectively, were o bserved after 28 days (Enriquez 1995). In fresh water, human enteroviruses can survive for several weeks. For instance, coxsackievirus B3, echovirus 7 and 9 poliovirus 1 can be inactivated by 6.5 - 7 logs over 8 weeks at 22 ° C , and 4 - 5 logs over 12 weeks at 1 ° C (Hurst 1989). In groundwater, the presence of indigen ous microorganisms is the important feature in inactivation of enteroviruses (Gordon and Toze 2003). Since UV is destructive for viruses, exposure to UV light or sunlight can enhance virus inactivation in the environment. For example, to achieve inactivati on rate of 99% for poliovirus without UV light in marine water, 52 days were needed, while in the presence of sunlight only 21 days w ere required (Rzezutka and Cook 2004). 1. 2.5. Virus t ransport in the e nvironment Batch experiments have been used to inve stigate the factors affecting virus - soil sorption behavior. Jin and Flury (2002) summarized the batch studies done over the previous 20 years. Bacteriophage indicators, and in some cases enteroviruses, were used, and most such studies focused on the effect of pH and ionic strength of the solution, the presence of compounds that compete for binding sites, isoelectric point (IEP) and hydrophobicity of the bacteriophage, and properties of the sorbent. The sorbents used in these studies were mostly soil (sand, silt and clay) and activated carbon. The Freundlich isotherm model ( where, C S is the quantity of virus sorbed per unit mass of soil; C L is the concentration of virus remaining in the liquid phase; K F is the Freundlich constant; 1/n is a constant) has been used to describe sorption (Drewry and Eliassen 1968; Bitton et al. 1976; Burge and Enkiri 1978; Gerba and Lance 1978; Moore et al. 1981; Jin et al. 1997; Bales et al. 1991; Powelson and Gerba 1994; Thompson et al. 1998; Powell et a l. 2000), and studies have determined that (i) clayey soils have higher virus sorption capacity, (ii) an increase in cation concentration in solution can increase virus sorption and (iii) pH affects virus sorption. Burge and Enkiri (1978) found a negative correlation between virus and soil pH, since the virus particles were more positively charged when the soil pH was low, and more readily sorbed on negatively charged soil 10 surfaces. The presence of organic matter (OM) enhances virus transport (Bixby et al . 1979; Moore et al . 1981; Fuhs et al . 1985; Powelson et al . 1991; Pieper et al . 1997; Zhuang and Jin 2003; Bradford et al . 2006) by competing with virus particles for binding sites and thickening the electrical double layer on sorbent and the virus particle s (Cao et al. 2010). Virus size and surface properties, such as isoelectric point (IEP) and hydrophobicity, play major roles in controlling virus sorption and transport. The size and IEP of selected viruses are summarized in Table 1 - 2. IEP is the pH at wh ich the virus particle has a net neutral charge. Virus particles exhibit a positive charge when the pH of a solution is below the IEP of virus, and a net negative charge at pH greater than IEP (Vega 2006). IEP has been suggested as the dominant factor cont rolling virus adsorption during transport through sandy soils (Dowd et al. 1998). However, Dowd et al. (1998) also found that isoelectric points of bacteriophage larger than 60nm did not affect sorption to soil and that bacteriophage size was the overridin g determinant of virus sorption. Zerda et al. (1985) observed that all viruses adsorbed to negatively charged surfaces at pH less than their respective IEP, while viruses would exclusively adsorb to positively charged surfaces at pH greater than IEP. When pH was close to the IPE, viruses adsorbed to all types of silica, although to a lesser extent. Herath et al. (1999) reported that the highest Nwachcuku and Gerba (2004) sug gested that low IEP typically makes microorganisms resistant to water treatment. Other parameters that control virus sorption and transport are zeta potential and hydrophobicity e closest separation between a small ion and the ch 2004), and it is related to the stability of colloidal dispersions. The zeta potential is a function of solution pH since viruses become more negatively charged in higher pH waters (Liu et al. 2009; Gitis et al . 2002). Ionic strength can also affect zeta potential. It has been reported that in 11 NaHCO 3 - NaCl solution (pH = 7), the zeta potential of poliovirus is - 1.8±0.3mV and - 5.9±0.9mV at ionic strengths of 0.3M and 0.2M, resp ectively (Murray and Parks 1980). At low zeta potentials, viruses tend to coagulate or flocculate, and thus their transport may be retarded. Hydrophobicity is another important surface property. It has been suggested that viruses with a lipid envelope are generally hydr ophobic, while viruses without a lipid envelope tend to be hydrophilic (Vidaver et al. 1973). Kinoshita et al. (1993) compared PRD - 1 and MS2 phages and suggested that the less hydrophilic phage (MS - 2) acted conservatively and was no t removed in sand columns at pH 5.7 - 8.0. Farrah et al. (1981) reported that hydrophobic interactions are the dominant determinant of virus attachment during flow through porous media so that hydrophobic effects are of primary importance to virus removal fro m water (Powelson 1990; Murray 1980). Numerous studies have used isotherm approaches to evaluate the factors that affect desorption behavior of chemical compounds, but desorption isotherms have not been developed for viruses or viral indicators. Chetochine et al . (2006) found that after a series of 17 extractions (25ml sample volume with 2% biosolids) from solid media, 10 3 PFU of bacteriophage MS2 remained in the pelletized solid, but almost no MS2 were in the supernatant. Also, it has been rep orted that enteroviru ses (Gerba 1981; Pancorbo et al . 1981) and coliphage (Gerba et al . 1978) attach strongly to solid phases and are difficult to elute from sludge. 1. 3. Detection Metnods Traditionally, cell culture has been the method used for virus detection. In this meth od, infected cell cultures undergo morphological changes called cytopathic effects (CPEs) that are observed microscopically. The method is labor intensive and some viruses do not exhibit 12 CPEs. Also traditionally, plaque assays are used to detect phages. In this method, a confluent monolayer of host cells is infected with the virus, and the infected area will create a plaque. By counting the number of plaques, virus concentration can be determined and represented in terms of plaque forming units. PCR is emer ging very rapidly as a method for virus detection in environmental samples. Compared to cell culture, the main advantages of PCR methods for virus detection include fast results, high specificity and sensitivity, and the ability to detect difficult to cult ure or non - culturable viruses such as adenovirus 40/41 and noroviruses. The main disadvantage of PCR methods is that they do not provide a measure of infectivity. There are also problems associated with detection limits and environmental inhibition. Microa rrays can also be used for the detection of viruses. Hundreds or thousands of genes can be studied simultaneously using DNA microarrays, and the procedure is relatively fast. Conventional PCR can amplify and detect virus - specific DNA sequences in the prese nce of DNA from many other sources. Gel electrophoresis is needed afterward in order to visualize the results. Normally conventional PCR is not a quantitative assay, but quantitative results can be generated by using dilutions and the most probable number (MPN) method. Reverse transcription PCR is used to produce a complementary strand (cDNA) for RNA viruses such as enteroviruses and noroviruses. Nested PCR generally has two sets of primers, one set nested within the nucleic acid defined by the second prime r pair. An amplicon is generated by the outer primers, while the target sequence of DNA is amplified by inner primers. In Multiplex PCR, multiple DNA sequences are targeted simultaneously. Real - time PCR is a quantitative assay in which target sequences are simultaneously amplified and quantified. In addition to primers, a set of probes with attached dyes is involved in real - time PCR. During amplification, the dyes are released from the probes and fluoresce. The fluorescence signal can be detected and, using a standard curve, the number of viral genome copies is quantified. When combined with cell culture, PCR can be employed to determine 13 the infectivity of viruses using a procedure called integrated cell culture PCR (ICC - PCR). A simplified schematic of virus detection methods in environmental media is shown in Figure 1 - 2. Sample collection and pre - treatment is a critical aspect of all environmental virology methods and pre - treatment methods are also shown in Figure 1 - 2. Virus concentration in natural water bo dies is usually low, and pre - concentration of viruses is often the most important step for effective detection. The technique most commonly used to concentrate viruses from water samples is the virus adsorption - elution microporous filter method, or VIRADEL . The filters for VIRADEL can be electropositive or electronegative. When using negative filters, adjustment of cationic salt concentration and pH is needed prior to sample processing. Electropositive filters do not require pre - treatment. The most commonly used electropositive filters are 1MDS filters and NanoCeram cartridge filters. After filtration, an elution step follows. The purpose of elution is to release the viruses captured by the cartridge filters (water samples) or to isolate viruses from sludg e/sediment grab samples. The adsorptio n - elution VIRADEL method (USEPA 2001a). Briefly, the filters are backwashed with beef extract solution. Elutes containing viruses are flocculated by lo wering pH. Flocs are isolated by centrifugation and re - suspended in sodium phosphate. Following neutralization and centrifugation, supernatants containing viruses are separated. Sludge, sediment or biosolids samples for viral analysis are eluted using ASTM Method D4994 - 89. The samples are mixed with beef extract, and pH is adjusted to about 3.5 to promote flocculation. Pellets are collected after centrifugation and re - suspended in phosphate buffered saline. pH is neutralized, before eluted samples are passe d through membrane filters. 14 1. 4. Fate of Viruses during Water Treatment 1. 4.1. Fate of v iruses during f ull - s cale w ater t reatment Since enteric viruses are transmitted mostly by the fecal oral route, water treatment provides a critical barrier to the re lease of viruses in potable water. According to the EPA National Primary Drinking Water Standards, enteric viruses must be re moved or inactivated by 4 logs (99.99%) during water treat ment from surface waters (USEPA 2001b). However, on the several occasions , viruses have been released by drinking water utilities. In general, even though most water treatment plants can achieve more than 4 log virus reduction (Payment et al. 1993, Paymet et al. 1985), viruses have been detected in finished water. A possible ex planation for those observations lies in the susceptibility of viruses to chlorine inactivation (Payment et al. 1985). Coxsackieviruses are more resistant to chlorination than polioviruses or reoviruses. To achieve 4 - log inactivation for coxsackieviruses, 40 minutes contact time is generally needed compared to 5 minutes for reoviruses (Payment et al. 1985). Virus survival in finished water also results from operational difficulties that lead to violation of treatment objectives related to turbidity and chlo rine residual. Inadequate floc formation, floc breakdown, and filter overloading can lead to ineffective disinfection and virus survival (Keswick et al. 1984). For example, Keswick et al. (1984) detected rotaviruses or enteroviruses in effluent from a conv entional drinking water treatment plant. They reported that 25 - 93% enteric viruses were removed during the dry season, while the removal efficiency was only 0 - 43% during the rainy season. When the quality of water declined, the removal of viruses decre ased as well. One of the possible reasons was adsorption to the particles that were not removed during clarification and filtration, and protected the viruses from final chlorination. 15 1. 4.2. Virus i nactivation Commonly used methods for drinking water d isinfection are chlorination, ozonation, and UV irradiation. Chlorine achieves inactivation/destruction by oxidizing cellular materials of target microorganisms. This technique is cheap and well - established, but carcinogenic chlorination by - products may be formed under certain conditions (USEPA 1999b). Chlorine dose and contact time are keys to virus removal. Higher dose and longer contact time generally produce higher removal efficiencies. For example, Abad et al. (1994) reported that the log inactivation of adenoviruses rose from 2.5 to 3.2 by doubling the dose of free chlorine. Shin and Sobsey (2008) reported that inactivation of poliovirus was enhanced with higher dose of chlorine, even though the contact time was shorter. A series of experiments carried out by Thurston - Enriquez et al. (2003a) showed that the virus removal (adenovirus 40 and poliovirus 1) was directly related to contact time. Similar results were obtained by Thurston - Enriquez et al. (2005b) using chlorine dioxide. The pH for disinfection usually removal efficiencies for polioviruses can be obtained at pH 10 compared to pH 6, but the effect of pH on virus inactivation during disinfection remains unce rtain and may vary between viruses. Ozone is more effective than chlorine for virus disinfection but provides no residual for protection against regrowth during water distribution. It is also very reactive and corrosive, and the cost of ozonation can be high. In addition, the presence of bromide ion in the raw water may lead to formation o f brominated by - products (USEPA 1999b). The mechanism of ozone disinfection involves destruction of the cell structures (cell wall, nucleic acids, etc.) by direct oxidat ion or reactions involving radical intermediates that are produced du ring ozone decomposition (USEPA 1999b; USEPA 1999c). Similar to chlorination, higher dose of ozone and longer contact time generally result in better performance for virus inactivation. F or instance, the log removal of poliovirus doubled when the ozone dose increased from 0.4mg/l 16 to 1.24mg/l (Katzenelson et al. 1979), while adenovirus removal slightly increased as a consequence of longer contact time (Thurston - Enriquez et al. 2005a). Tempe rature seems to be another important parameter, and lower temperature tended to facilitate virus inactivation (Herbold et al. 1989). No uniform relationship was found between pH and inactivation efficiency. UV irradiation can penetrate cell structures, da mage genetic materials and interfere with cell reproduction. It involves no chemical addition, and thus no residual or chemical intermediates will be formed and released to the environment. Disinfection with UV may depend on UV lamp type. For instance, med ium - pressure UV lamps can achieve higher inactivation rates compared to low - pressure lamps at the same total intensity (Eischeid et al. 2009, Guo et al. 2010, Linden et al. 2007; Linden et al. 2009). Higher UV dose can steadily increase inactivation of a v ariety of viruses, such as echovirus, coxsackievirus, poliovirus, and adenovirus (Gerba et al. 2002b; Ko et al. 2005; Thompson et al. 2003; Simonet and Gantzer UV dose is low. For example, it is widely known that human adenoviruses are very resist ant to UV (Ballester and Malley 2004; Chang et al. 1985; Eischeid et al. 2009; Gerba et al. 2002b; Ko et al. 2005; Nwachuku et al. 2005; Thurston - Enriquez et al. 2003b). 1. 5. Fat e of Viruses in Wastewater Treatment Systems 1. 5.1. Virus removal in f ull - s cale wastewater u tilities Wastewater is a primary source of human viruses in the environment. Conventional full - scale wastewater treatment utilities release infectious and non - infec tious viruses in their effluent (Katayama et al. 2008; Haramoto et al. 2007; Hewitt et al. 2011; Petrinca et al. 2009; Aulicino et al. 1996; Costan - Longrades et al. 2008; Lodder et al. 2005; Rose et al. 1996; Nordgren et al. 2009; Haramoto et al. 2007; Kit ajima et al. 2009; Payment et al. 2001; Prado 17 et al. 2011; Simmons and Xagoraraki 2011). Membrane bioreactors (MBRs) are expected to provide higher quality effluents. This technology involves the combination of the activated sludge biological treatment wit h biomass separation by membrane filtration in a submerged or side - stream configuration. When well designed and operated, MBRs can consistently achieve efficient removals of suspended solids (Vaid et al. 1991), chemical oxygen demand (Pankhania et al. 1994 ; Beaubien et al. 1996), biochemical oxygen demand (Kishino et al. 1996), nitrogen (Kishino et al. 1996; Gujer et al. 1999), phosphorus (Schaum et al. 2005) and coliform bacteria (Van der Roest et al. 2 002). Under optimal conditions, MBR systems can also r eliably remove various viruses and phages (Table 1 - 3). For example, Kuo et al. (2010) reported 4.1 - 5.6 log removals for human adenoviruses, while Simmons et al. (2011) reported that removal efficiencies could reach 6.3, 6.8, and 4.8 logs for human adenovir uses, enteroviruses, and noroviruses respectively. Da Silva et al. (2007) obtained high removal efficiencies for noroviruses in a full - scale MBR system, but their data also suggest that virus removals were inconsistent. Removal of viruses in full - scale con ventional wastewater treatment plants (WWTP) and full - scale MBR systems are compared in Figures 1 - 3 through 1 - 6. Overall, full - scale MBR plants achieved higher virus removals. Adenovirus removal in WWTPs prior to disinfection (Table 1 - 3 and Figure 1 - 3) ran ged from 1.02 logs to 4.08 logs (Haramoto et al. 2007; Hewitt et al. 2011). Katayama et al. (2008) reported that in WWTPs, the virus removal due to disinfection was 1.65 logs on average. Adenovirus removals in advanced treatment systems such as MBRs were s ignificantly higher ranging from 3.4 logs to 6.3 logs (Ku o et al. 2010; Simmons et al. 2011; Simmons and Xagoraraki 2011). Figure 1 - 4 shows a summary of enterovirus removals in full - scale WWTPs. In conventionally treated wastewater prior to disinfection , virus removals ranged from 0.7 logs to 2.4 logs (Lodder et al. 2005 ; Costan - Longrades et al. 2008 ; Hewitt et al. 2011). Conventional plants with disinfection produced higher virus removals: up to 5.23 logs 18 (Aulicino et al. 1996 ; Petrinc a et al. 2009; Kat ayama et al. 2008 ; Costan - Longrades et al. 2008 ; Rose et al. 1996). MBR plants without disinfection removed enteroviruses from 4.1 to 6.8 logs (Simmons et al. 2011). As shown in Figure 1 - 5, reduction of norovirus I in conventional WWTPs without disinfecti on was less than 1.4 logs (Hewi tt et al. 2011 ; Nordgren et al. 2009). WWTPs with disinfection performed slightly better with log removals from 0.95 to 2.69 (Katayama et al. 2008). In MBR plants without disinfection the removal of norovirus I was up to 5.5 logs (da Silva et al. 2007). Norovirus II removals in full - scale WWTPs are summarized in Figure 1 - 6. The highest virus reduction in a conventional WWTP without disinfection was 1.2 logs (Hewitt et al. 2011 ; Nordgren et al. 2009), whereas, with disinfection , virus removal ranged from 1.3 to 3 logs (Katayama et al. 2008). For MBR plants, removals in the range of 2.3 logs to 4.9 logs were observed (da Silva et al. 2007 ; Simmons et al. 2011). 1. 5 .2. Virus r emovals in b ench and p ilot - scale MBR s ystems Bench a nd pilot - scale MBR studies have been performed to describe virus removal. MS - 2 coliphage appears to be the most common virus used in bench scale MBR studies. It is a single - stranded RNA virus, with icosahedral shape, small size (20 nm to 25 nm), and low IE P (3.9) (Zerda 1982) and relative hydrophobicity (Oh et al. 2007). These characteristics are similar to some pathogenic human viruses found in water and wastewater such as hepatitis A virus and poliovirus (Fiksdal et al. 2006), and thus make MS - 2 a good in dicator and surrogate for virus studies with membrane systems (Shang et al. 2005; Comerton et al. 2005). Both indigenous and lab - cultured MS - 2 phages were used in these studies, and quantification was done by plaque assay. T4 coliphage has also been used i n bench - scale MBR studies since it is similar to adenoviruses, reoviru ses, rotaviruses (Zheng and Liu 2007), and coronaviruses (Lv et al. 2006). Even though the size and IEP of phages are similar to those of some enteric 19 viruses, their removal and transpor t do not necessarily relate to those of enteric viruses in wastewater systems, and therefore further research is needed. As shown in Table 1 - 4, bench and pilot scale MBRs can achieve high removals of coliphages. Five potential mechanisms for virus removal were suggested (Ravindran et al. 2009): (1) rejection of virus by a gel layer consisting of natural organic matter; (2) rejection by a layer of microbial biomass; (3) rejection due to internal pore blocking by natural organic matter; (4) adsorption on the surface of membranes and bio - particles; and (5) combinations of these mechanisms. MBR systems with higher hydraulic retention times (HRT) and lower solids retention times (SRT) appear to be more efficient in removing viruses (Wu et al. 2010). Madaeni et al . (1995) suggested that the presence of biomass, low trans - membrane pressure and stirring enhance virus removal during the membrane filtration process. Membrane pore size may be an important determinant of virus removal efficiency. Membranes with smaller pore sizes tend to achieve higher removal for viruses, but not always (Figure 1 - 7). Madaeni et al. (1995) reported that hydrophobic PVDF membrane (pore size = 0.22 µm) could remove about 99% of poliovirus, while ultrafiltration membranes with pore sizes sm aller than the virus achieved complete rejection. However, it has been observed that in MBR systems with a range of membrane pore sizes (0.03 - 0.1 µm) indigenous MS - 2 was not detectable in the effluent, and removal mechanisms other than straining may exist (Hirani et al. 2010). According to Zheng and Liu (2007) and Zheng et al. (2005), there was no significant difference in virus removal efficiency using membranes with 0.1 µm and 0.22 µm pore sizes, whereas Lv et al. (2006) indicated that a 0.1 um membrane w as more effective than a comparable 0.22 µm membrane. Fiksdal et al. (2006) reported that phages were poorly removed during MBR treatment without pre - coagulation / flocculation, even using ultrafiltration membranes. 20 1. 5 .3. Viruses in b iosolids Most waste water virus studies report numbers of viruses in effluent or removal efficiencies that reflect virus concentrations in influent and effluent. Since viruses tend to attach to solid surfaces, most viruses that survive wastewater treatment are likely associat ed with waste activated sludge and may be present in biosolids. In the US, approximately 5.6 million dry tons of biosolids are generated annually, 60 percent of which are land applied as a soil amendment (NRC 2002). The US EPA divides biosolids into two cl asses: class A or pathogen - free biosolids, and class B biosolids, which may have some pathogens such as human adenovirus (USEPA 2003). Different treatment methods can be used to produce class A biosolids, and the removal of viruses is established using bac terial indicators such as fecal coliforms (USEPA 2003). Class A biosolids are sold directly to the public for lawn and garden use and should not contain detectable pathogens. Class B biosolids can be applied on agricultural and forest lands as fertilizers. Monitoring for enteroviruses in biosolids is now encouraged but not required by the EPA, and reports of enteric viruses in sludge and biosolids are limited. Table 1 - 5 indicates that class B biosolids contain potentially infectious viruses. Using integrate d cell culture - PCR, relatively large numbers of viable viruses have been detected in class B biosolids (Wong et al. 2010). 1. 5 .4. Bacterial v iruses ( p hages) in w astewater Bacteriophages, or phages, are viruses that infect bacteria. All contain nucleic ac id surrounded by a protein coat that enables them to stick to bacterial cell envelopes. When attached, they inject DNA into the host bacteria. It is suggested that phage abundance in activated slud ge at wastewater treatment plants is higher than any other environment (Shapiro et al. 2011; Rosenberg et al. 2010; Wu and Liu 2009; Otawa et al. 2007). In activated sludge the phage - to - bacterial - cell - ratio is approximately 10:1 (Rosenberg et al. 2010). Thus, 21 important phage - bacteria interactions may take place du ring wastewater treatment. For example, bacteriophages may play a major role in bacterial evolution by facilitating the transfer of antibiotic resistance genes (ARG) or other genes to new bacterial hosts via transduction (Mazaheri Nezhad Fard et al. 2010; Canchaya et al. 2004; Boyd and Brussow 2002). Horizontal gene transfer is the movement of genetic material among bacterial species without cell division. It provides an important mechanism for accelerating the dispersal of ARGs in the environment (Colomer - Lluch et al. 2011; Baquero et al. 2008; Sander et al. 2001). In recent years, there have been many efforts to study gene transfer mechanisms that are responsible for the spread of antibiotic resistance among bacteria. Transformation is the direct uptake of naked DNA from the cell surroundings. Conjugation is the transfer of DNA mediated by a conjugative or mobilizable genetic element (plasmids or transposons). It requires cell to cell contact and long fragments of DNA can be transferred through this mechani sm. The transfer of DNA mediated by bacteriophage is known as transduction. Very little information is available regarding phage - mediated transduction (Colomer - Lluch et al. 2011; Sander et al. 2001). Only a small fraction of general transducing bacteriopha ges have been characterized so far, and only a few studies have looked for antibiotic resistance genes in bacteriophage isolated from wastewater treatment plants or surface waters impacted by the discharge of treated wastewater (Colomer - Lluch et al. 2011; Mazaheri Nezhad Fard et al. 2010; Parsley et al. 2010; Muniesa et al. 2004; Prescott 2004). For example, Colomer - Lluch urban sewage and river water samples and fou nd that phages may act as reservoirs for the spread of ARGs in the environment. Another study was done on enterococcal bacteriophages that play a role in successful transfer of antibiotic resistant genes for tetracycline and gentamicin resistances between the same and different enterococcal species (Mazaheri Nezhad Fard et al. 2011). There are other ways in which bacteriophages are important in wastewater treatment 22 systems. As mentioned previously, bacteriophages infect bacteria; thus, they can control bac terial community structure. Researchers have proposed the use of phages during wastewater treatment to improve effluent and sludge characteristics (Withey et al. 2005). Using phages, it may be possible to improve wastewater treatment performance by, for ex ample, controlling foam in activated sludge treatment, attacking pathogenic bacteria, and reducing the competition between insignificant (from the perspective of waste conversion) and critically important bacterial populations. However, such modifications require a more complete understanding of wastewater microbial community dynamics including phage - dependent interactions (Withey et al. 2005). Next generation sequencing and metagenomics are powerful tools that can provide information about phages and their significance. 1. 6 . Viral Risk Assessment Quantitative viral risk assessment (QVRA) studies have been published for wastewater systems. Exposure to human enteric viruses from wastewater - related products (post - disinfected effluents and sludge) occur duri ng recreational activities in surface waters, sludge handling, land application of biosolids, ingestion of untreated surface and ground waters and other exposure pathways resulting in inhalation and inges tion - related health risks (Haas 1983; Lapen et al. 2 008; Viau and Peccia 2009 ). In general, quantitative microbial risk assessment includes hazard identification, exposure assessment (determination of exposure routes, pathogen dose, and exposure parameters), determination of dose - response relationships, an d risk characterization. Dose - response assessment characterizes the correlation between probability of infection and exposure to viruses. The number of viruses ingested is estimated by Equation 1 (Haas et al. 1999). The exponential model (Equation 2) and b eta - Poisson model (Equation 3) have been 23 used extensively to represent dose - response relationships (Table 1 - 6) and estimate the - Poisson model approaches the exponential model (Haas et al. 1999). Equation [1] Equation [2] Equation [3] Where, N is number of viruses ingested; C is the concentration of viruses; R is the efficiency of recovery method; I is the fraction of detected viruses that are capable of infection; DR is the removal or inactivation efficiency of the treatment process. For recreational water , DR is equal to 0, since no treatment is applied; V is the daily volume of recreational water consumed by individu als that are exposed to the water. P i/day is the Several QVRA studies have been performed using virus indicators such as bacteriophage and viruses in the environmen t (Ha as 1983; Regli et al. 1991; Dowd et al. 2000; Gerba et al. 2002a; Eisenberg et al. 2006 and 2008). QVRA studies have generally used culture - based virus measurements to estimate ingested viral dose, assuming that a single virus can be used to represent tota l human enteric viruses (Haas 1983; Regli et al. 1991; Gerba et al. 2002a; Eisenberg e t al. 2008). For example, during biosolids - based QVRA studies (Gerba et al. 2002a; Eisenberg et al. 2008) the total concentration of biosolids - associated viruses was repr esented in terms of the measured concentrations of rotaviruses or echovirus - 12 to calculate risk estimates. Other QVRA studies have used viral genomic copies (GCs) measured via PCR to estimate ingested dose of a specific virus type, with or without adjustm ents to convert GCs to infectious virus concentrations (Masago et al. 2006; Teunis et al. 2008; Schoen and Ashbolt 2010). Masago et al. (2006) assumed that the total GC measurement of noroviruses represents the infectious concentration of noroviruses to as sess 24 risk from ingestion of water. Teunis et al. (2008) and Schoen and Ashbolt (2010) assumed that the infectious concentration of noroviruses is half the measured number of norovirus GCs in order to estimate the risk of infection from ingestion of water d uring recreational activities. Viau and Peccia (2009) used a similar approach for converting adenovirus GCs to infectious adenovirus concentration for estimating risks of inhalation of bioaerosols (0.1% conversion factor calculated using data for primary e ffluent samples obtained fr om He and Jiang (2005)) . The use of different assumptions for relating GCs to infectious virus concentrations (infectivity ratios) in QVRA studies poses a consistent and significant uncertainty in estimates of infectious viral do ses. The risk of virus infection from applied biosolids appears to be low. For example, Gerba et al. (2002a) estimated that such risk was less than 10 - 4 (1 out of 10,000 risk of infection). Kumar et al. (2012) reported that the viral infection risk of soi l ingestion of biosolids was greater than 10 - 4 , based on the data obtained from both cell culture and genomic methods. At recreational beaches, Wong et al. (2009a) estimated the daily risk of viral infection ranged from 0.2 to 2.4 per 1000 swimmers. 1. 7 . Summary and Conclusions Occurrence of human pathogenic viruses in environmental waters (i.e. surface waters, groundwater, drinking water, recreational water, and wastewater) raises concerns regarding the possibility of human exposure and waterborne infecti ons. Commonly observed waterborne viruses include adenoviruses, enteroviruses, noroviruses, and rotaviruses. Much attention has been given recently to human adenoviruses due to related health implications that range from diarrhea to death. Viruses are the smallest of all microorganisms, and their size facilitates transport in environmental media. In addition, viruses have very low die - off rates and low infectivity 25 doses, increasing concern over outbreaks of disease related to waterborne or sludge - related v irus exposures. The ability to detect waterborne viruses effectively is the basis for microbial risk assessment and management of water resources for the protection of public health. However, precise detection, quantification, and infectivity determination for viruses remain challenging. Wastewater is a major source of viruses in the environment. Especially when water reuse is contemplated, appropriate technologies must be practiced that yield a virus - free effluent. Membrane bioreactors have been shown to r educe numbers of viruses more effectively than conventional activated sludge facilities. Even though advances in wastewater treatment technology in recent decades have greatly reduced waterborne disease, human enteric viruses are still detected in the effl uents of state - of - the - art wastewater treatment plants worldwide, including those with membrane bioreactors. Viruses have also been observed in the effluent of conventional drinking water utilities. In drinking water treatment, inactivation of resistant vi ruses poses a challenge, particularly for small - scale or point - of - use systems. For example, adenoviruses are very resistant to UV disinfection. Overall, the presence of viruses in water and wastewater is a difficult problem for environmental engineers, d ue to the small sizes, prevalence, infectivity, and resistance of viruses to disinfection. Here, we briefly described virus survival and behavior in the environment and reviewed both virus - associated diseases and their transmission pathways. Environmental engineers should be aware that wastewater treatment plants are not able to remove many viruses from wastewater. Viruses discharged from drinking water treatment plants due to technical and management deficiencies may increase human exposure and disease. Th e knowledge summarized provides basic information needed to make decisions for efficient water and wastewater management and reduction of risk arising from human exposure to viruses. 26 APPENDIX 27 Table 1 - 1. Human Viruses in the Environmental Prote ction Agency Contaminant Candidate Lists (CCL) Virus Family Classification CCL 1 CCL 2 CCL 3 Adenoviruses Adenoviridae Group I (double strand DNA) Yes Yes Yes Enteroviruses* Picornaviridae Group IV (positive single - stranded RNA) --- --- Yes Coxsackievi ruses Yes Yes --- Echoviruses Yes Yes --- Hepatitis A viruses --- --- Yes Caliciviruses Caliciviridae Group IV (positive single - stranded RNA) Yes Yes Yes * Polioviruses, coxsackieviruses, and echoviruses are generally referred to as enteroviruses . 28 Table 1 - 2. Summary of Virus Surface Properties Affecting Sorptive Removal from Water Virus (1) Virion Size (nm) Isoelectric Point (IEP) References Enterovirus 22 - 30 4.0 - 6.4 Minor, 1987; Grce and Pavelic, 2004; Murry and Parks, 1980; Butler et al., 1985; Zerda and Gerba, 1984 Coxsackieviruses 4.75 - 6.75 Echoviruses 4.0 - 6.4 Hepatitis A viruses 27 - 28 2.8 Minor, 1987; Nasser et al., 1992 Caliciviruses 30 - 40 5.5 - 6.0 (2) Carter et al., 1987; Goodridge et al., 2004 Adenoviruses 70 - 140 3.5 - 4.5 Nermut, 1987; Trilisky and Lenhoff, 2007; Wong et al., 2012b; Stewart, 1991 (1) All viruses in CCL are non - enveloped and i cosahedral in shape. (2) For Norwalk virus (a member of norovirus es ). 29 Table 1 - 3. Virus Removal in Full - Scale Me mbrane Bioreactors Membrane pore size Virus (source) Detection methods Removal efficiency Reference 0.4um F - specific Coliphage Plaque assay 6.0 logs Zanetti et al., 2010 0.4 um Somatic Coliphage Plaque assay 4.0 logs Zanetti et al., 2010 0.1 um HAdV qP CR 4.1 - 5.6 logs Kuo et al., 2010 0.4 um Norovirus I Norovirus II qPCR 0 - 5.3 logs* 0 - 5.5 logs* da Silva et al., 2007 NA HAdV Enterovirus qPCR 3.4 - 4.5 logs* 2.9 - 4.6 logs* Simmons and Xagoraraki, 2011 0.1 um HAdV Enterovirus Norovirus (II) qPCR 4.1 - 6.3 logs 4.1 - 6.8 logs 3.5 - 4.8 logs Simmons et al., 2011 * Obtained from graphs 30 Ta ble 1 - 4. Virus removal in bench and pilot - scale membrane bioreactors Scale Membrane pore size Virus (source) Detection methods Removal efficiency Reference Bench 0. 4 µm MS - 2 Plaque assay 0.4 - 2.5 logs Shang et al., 2005 Bench 0.2 µm MS - 2 Plaque assay Average 6.7 logs Fiksdal et al., 2006 Bench 0.45 µm MS - 2 Plaque assay 0.31 - 1.5 logs Oh et al., 2007 Bench UF and NF MS - 2 Plaque assay 2 logs for UF 4 logs for NF Hu et al., 2003 Bench 0.1 and 0.22µm T4 Coliphage Plaque assay 5 - 8 logs for 0.1 um 3.5 - 6 logs for 0.22um Lv et al., 2006 Bench 0.1 and 0.22µm T4 Coliphage Plaque assay 5.5 logs Zheng and Liu., 2007 Bench 0.1 and 0.22µm T4 Coliphage Plaque assay 6 logs Zheng et al., 2005 Bench 0.4µm Somatic Coliphage Plaque assay 1.5 - 2.5 logs Wu et al., 2010 Pilot 300k Da MS - 2 Plaque assay No plaques observed Cicek et al., 1998 Pilot 0.04 - 0.1 µm MS - 2 Plaque assay 1.0 - 4.4 logs Hirani et al., 2010 Pilot 0.2 µm MS - 2 Plaque assay 3.8 logs Ravindran et al., 2009 Pilot 0.1 µm Somatic Coliphage Plaque assay No plaques observed Ahn et al., 2001 Pilot 0.03 µm Somatic Coliphage Plaque assay 3.7 logs Wong et al., 2009b Pilot 0.4 µm F - specific Coliphage Plaque assay > 4.0 logs Tam et al., 2007 Pilot 0.1µm F - specific Coliphage Plaque assay No plaques observed Ahn et al., 2001 Pilot 0.04 µm Enteric cytopathogenic bovine orphan virus Plaque assay Not detectable in effluent Krauth et al., 1993 Pilot 0.45 µm Norovirus E nterovirus PCR - 0.19 - - 0.01 - 0.05 - - 0.03 Ottoson et al., 2006 31 Table 1 - 5. Virus occurrence in dewatered sludge and class B biosolids Author Detection method Viruses Occurrence average Dewatered Sludge Bofill - Mas et al., 2006 qPCR Adenoviruses 1.1 × 1 0 2 copies/g Monpoeho et al., 2001 RT - PCR Enteroviruses 4.8 × 10 4 copies/10g Cell Culture 7 MPNCU*/10g Viau and Peccia, 2009 qPCR Adenoviruses 2.5 × 10 4 copies/g Wong et al., 2010 qPCR Adenoviruses 1.9 × 10 8 copies/g Enteroviruses 2.3 × 10 5 copies/ g Cell Culture Adenoviruses 2210 MPN/4 g Enteroviruses Class B Biosolids Bofill - Mas et al., 2006 qPCR Adenoviruses 10 3 copies/g Monpoeho et al., 2001 RT - PCR Enteroviruse s 1.06 × 10 4 copies/10g Cell Culture 9 MPNCU/10g Monpoeho et al., 2004 RT - PCR Enteroviruses 1.2 × 10 4 copies/g Cell Culture 38.2 MPNCU/g Viau and Peccia, 2009 qPCR Adenoviruses 5 × 10 5 copies/g Wong et al., 2010 qPCR Adenoviruses 7.5 × 10 5 copies/g Enteroviruses 1.9 × 10 4 copies/g Norovirus GI 5 × 10 4 copies/g Noro virus GII 1.5 × 10 5 copies/g Cell Culture Adenoviruses 480 MPN/4g Enteroviruses Norovirus GI Norovirus GII *MPNCU most - probable - number cytopathogenic units. 32 Table 1 - 6. Dose response models for enteric viruses Waterborne Virus Exposure Dose - response Model Defined Parameters Reference Enteroviruses 68 - 71 Ingestion Beta - Poisson Soller et al., 2004 Poliovirus 1 Ingestion Exponential r = 0.009102 Regli et al., 1991 Minor et al., 1981 Poliovirus 1 Ingestion Beta - Poisso n Regli et al., 1991 Lepow et al., 1962 Poliovirus 3 Ingestion Beta - Poisson Regli et al., 1991 Katz et al., 1967 Coxsackievirus A21 Inhalation Exponential r = 0.0145 Haas et al., 1999 Coxsackievirus B4 Exponen tial r = 0.007752 Haas et al., 1999 Echovirus 12 Ingestion Exponential r = 0.012771 Haas et al., 1999 Echovirus 12 Ingestion Beta - Poisson Regli et al., 1991 Schiff et al., 1984 Human adenovirus 4 Inhalation Exponential r = 0.4172 Haas et al., 1999 Mena and Gerba, 2009 Human caliciviruses Ingestion Beta - Poisson a =0.126 - 0.21 - 0.84 Soller et al., 2004 Noroviruses Ingestion Exponential r = 0.069 Masago et al., 2006 Rotavirus Ingestion Beta - Poisson Reg li et al., 1991 Haas et al., 1999 Ward et al., 1986 Teunis et al., 2008 Hepatitis A virus Ingestion Exponential r = 0.548576 Haas et al., 1999 33 Figure 1 - 1. Sources of viruses in the environment 34 Figure 1 - 2. Summary of virus elution and detecti on methods 35 Figure 1 - 3. Adenovirus removal in full - scale wastewater treatment plants 36 Figure 1 - 4. Enterovirus removal in full - scale waste water treatment plants 37 Figure 1 - 5. Norovirus I removal in full - scale waste water treatment plants 38 Figure 1 - 6. Norovirus II removal in full - scale waste water treatment plants 39 Figure 1 - 7. Virus removal as a function of membrane pore size in bench and pilot scale MBR systems 40 REFERENCES 41 R EFERENCES Abad, F. X., Pinto, R.M., Diez, J.M., Bosch, A. (1994). Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine. Applied and Environmental Microbiology, 60(7): 2377 - 2383. Abbaszadegan, M., LeChevallier, M., Gerba. C. (2003). Occurrence of Viruses in US Groundwaters. Journal American Water Works Association . 95(9):107 - 120. Abzug, M. J., Cloud, G., Bradley, J., et al. (2003). Double blind placebo - controlled trial of pleconaril in infants with enteroviral meningitis. The Pediatric Infectious Disease Journal , 22:335 340. Adrian, T., Wigand, R., Richter, J. (1987). Gastroenteritis in Infants Associated with Genome Type of Adenovirus 31 and with Combined Rotavirus and Adenovirus 31 Infection. European Journal of Pediatrics . 146: 38 - 40. Aggarwal, R., Krawczynski, K. (2000). Hepatitis E: an overview and recent advances in clinical and laboratory research. Journal of G astroenterology and H epatology , 15 (1), 9 - 20. Ahmed, W., Stewart, J., Powell, D., & Gardner, T. (2008). Evaluation of Bacteroides marke rs for the detection of human faecal pollution. Letters in A pplied M icrobiology , 46 (2), 237 - 242. Ahmed, W., Goonetilleke, A., & Gardner, T. (2010). Human and bovine adenoviruses for the detection of source - specific fecal pollution in coastal waters in Aust ralia. Water R esearch , 44 (16), 4662 - 4673. Ahn, K., Song, K., Yeom, I., & Park, K. (2001). Performance comparison of direct membrane separation and membrane bioreactor for domestic wastewater treatment and water reuse. Water Supply , 1 (5 - 6), 315 - 323. Albert, M. J. (1986). Enteric Adenovirus Brief Review. Archives of Virology , 88: 1 - 17. Alvarez, Maria E., and O'Brien, R.T. (1982). Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine . Applied and Environmental Microbiology , 44(5): 1064 - 1071. Anderson, E. J., & Weber, S. G. (2004). Rotavirus infection in adults. The Lancet I nfectious D iseases , 4 (2), 91 - 99. Aslan, A., Xagoraraki, I., Simmons, F. J., Rose, J. B., & Dorevitch, S. (2011). Occurrence of adenovirus and other enteric viruses in limite d contact freshwater recreational areas and bathing waters. Journal of A pplied M icrobiology , 111 (5), 1250 - 1261. ASTM Method D4994 - 89. Standard practice for recovery of viruses from wastewater sludges. 2002. Aulicino, F.A., Mastrantonio, A., Orsini, P., Bell ucci, C., Muscillo, M., Larosa, G. (1996). 42 Enteric viruses in a wastewater treatment plant in Rome. Water, A ir and S oil P ollution , 91: 327 - 334. Ausar, S. F., Foubert, T. R., Hudson, M. H., Vedvick, T. S., & Middaugh, C. R. (2006). Conformational stability and disassembly of Norwalk virus - like particles effect of pH and temperature. Journal of Biological Chemistry , 281 (28), 19478 - 19488. Bales, R. C., Hinkle, S. R., Kroeger, T. W., Stocking, K., Gerba, C. P. (1991). Bacteriophage adsorption during transport t hrough porous media: Chemical perturbations and reversibility. Environmental S cience & T echnology , 25 (12), 2088 - 2095. Ballester, N. A., Malley, J. P. (2004). Sequential disinfection of adenovirus type 2 with UV - chlorine - chloramine. Journal (American Water Works Association) , 96 (10), 97 - 103. Baquero, F., Martínez, J. L., Cantón, R. (2008). Antibiotics and antibiotic resistance in water environments. Current O pinion in B iotechnology , 19 (3), 260 - 265. Barwick, R. S., Levy, D. A., Craun, G. F., Beach, M. J., Cal deron, R. L. (2000). Surveillance for waterborne - disease outbreaks United States, 1997 1998. CDC Surveillance Summaries , 49 , 1 - 35. Beaubien, A., Baty, M., Jeannot, F., Francoeur, E., Manem, J. (1996). Design and operation of anaerobic membrane bioreactors: development of a filtration testing strategy. Journal of Membrane Science , 109 (2), 173 - 184. Bitton, G., Pancorbo, O., Gifford, G. E. (1976). Factors affecting the adsorption of polio virus to magnetite in water and wastewater. Water Research , 10 (11), 973 - 980. Bixby, R. L., O'Brien, D. J. (1979). Influence of fulvic acid on bacteriophage adsorption and complexation in soil. Applied and E nvironmental M icrobiology , 38 (5), 840 - 845. Blackburn, B. G., Craun, G. F., Yoder, J. S., Hill, V., Calderon, R. L., Chen, N., Lee, S. H., Levy, D. A., Beach, M. J. (2004). Surveillance for waterborne - disease outbreaks associated with drinking water --- United States, 2001 -- 2002. MMWR Surveill Summ , 53 (8), 23 - 45. Bofill - Mas, S., Albinana - Gimenez, N., Clemente - Casares, P., Hundes a, A., Rodriguez - Manzano, J., Allard, A., Calvo, M., Girones, R. (2006). Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices. Applied and E nvironmental M icrobiology , 72 (12), 7894 - 7896. Bon, F., Fascia P., Dauver gne M., Tenenbaum D., Planson H., Petion A.M., Pothier P., Kohli E. (1999). Prevalence of Group A Rotavirus, Human Calicivirus, Astrovirus, and Adenovirus Type 40 and 41 Infections among Children with Acute Gastroenteritis in Dijon, France. Journal of Clin ical Microbiology . 37 (9): 3055 - 3058. Borchardt, M. A., Chyou, P - H., DeVries, E. O., Belongia, E. A. (2003a). Septic system density and infectious diarrhea in a defined population of children. Environmental Health Perspectives 111:742 - 748. Borchardt, M. A. , Bertz, P. D., Spencer, S. K., Battigelli, D. A. (2003b). Incidence of enteric viruses in groundwater from household wells in Wisconsin. Applied and Environmental Microbiology , 69 (2), 1172 - 1180. 43 Bosch, A., Pintó, R., Abad, F. (2006). Survival and transport of enteric viruses in the environment. Viruses in Foods , 151 - 187. Boyd, E. F., Brüssow, H. (2002). Common themes among bacteriophage - encoded virulence factors and diversity among the bacteriophages involved. TRENDS in Microbiology , 10 (11), 521 - 529. Bradfor d, S. A., Tadassa, Y. F., Jin, Y. (2006). Transport of coliphage in the presence and absence of manure suspension. Journal of E nvironmental Q uality , 35 (5), 1692 - 1701. Brunkard, J. M., Ailes, E., Roberts, V. A., Hill, V., Hilborn, E. D., Craun, G. F., Rajas ingham, A., Kahler, A., Garrison, L., Hicks, L., Carpenter, J., Wade, T. J., Beach, M. J., Yoder, J. S. (2011). Surveillance for waterborne disease outbreaks associated with drinking water --- United States, 2007 -- 2008. MMWR Surveillance Summary , 60 (12), 38 - 68. Burge, W. D., Enkiri, N. K. (1978). Virus adsorption by five soils. Journal of Environmental Quality , 7 (1), 73 - 76. Butler, M., Medlen, A.R. and Taylor, G.R. (1985). Electrofocusing of viruses and sensitivity to disinfection. Water Sci ence and Technol og y 17, 201 210. Canchaya, C., Fournous, G., Brussow, H. (2004). The impact of prophages on bacterial chromosomes. Molecular Microbiology , 53, 9 18. Cao, H., Tsai, F. T. C., & Rusch, K. A. (2010). Salinity and soluble organic matter on virus sorption in sand and soil columns. Ground Water , 48 (1), 42 - 52. Carter, M. J. and Madeley, C. R., (1987). Animal virus structure, Chapter 7, Caliciviridae, edited by Nermut, M. V. and Steven, A. C. Perspectives in Medical Virology , Vol. 3. Elsevier Science Publishers, Biom edical Division. Castignolles, N., Petit, F., Mendel, I., Simon, L., Cattolico, L., Buffet - Janvresse, C. (1998). Detection of adenovirus in the waters of the Seine River estuary by nested - PCR. Molecular and Cellular P robes , 12 (3), 175 - 180. Chang, J. C., Os soff, S. F., Lobe, D. C., Dorfman, M. H., Dumais, C. M., Qualls, R. G., Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. Applied and Environmental Microbiology , 49 (6), 1361 - 1365. Chen, C. H., Hsu, B. M., & Wan, M. T. (2008 ). Detection of enteroviruses within brackish water from the Damshui River watershed, Taiwan. Journal of Environmental Engineering , 134 (6), 486 - 492. Cicek, N., Winnen, H., Suidan, M. T., Wrenn, B. E., Urbain, V., & Manem, J. (1998). Effectiveness of the me mbrane bioreactor in the biodegradation of high molecular weight compounds. Water Research , 32 (5), 1553 - 1563. Chetochine, A. S., Brusseau, M. L., Gerba, C. P., Pepper, I. L. (2006). Leaching of phage from class B biosolids and potential transport through s oil. Applied and E nvironmental M icrobiology , 72 (1), 665 - 671. Clayson, E. T., Snitbhan, R., Ngarmpochana, M., Vaughn, D. W., & Shrestha, M. P. (1996). Evidence that the hepatitis E virus (HEV) is a zoonotic virus: detection of natural 44 infections among swine , rats, and chickens in an area endemic for human disease. Enterically - transmitted hepatitis viruses. Tours, France, La Simarre , 329 - 335. Cliver, D. O., Moe, C. L. (2004). Prospects of Waterborne Viral Zoonoses. Waterborne Zoonoses: Identification, Causes and Control , Edited by J. A. Cotruvo, A. Dufour, G. Rees, J. Bartram, R. Carr, D. O. Cliver, G. F. Craun, R. Fayer, V. P. J. Gannon, WHO. Colomer - Lluch, M., Jofre, J., Muniesa, M. (2011). Antibiotic resistance genes in the bacteriophage DNA fraction of en vironmental samples. PLoS One , 6 (3), e17549. Colson, P., et al . (2010). Pig liver sausage as a source of hepatitis E virus transmission to humans. Journal of Infectious Diseases , 202 (6), 825 - 834. Comerton, A. M., Andrews, R. C., Bagley, D. M. (2005). Evalu ation of an MBR - RO system to produce high quality reuse water: Micirobial control, DBP formation and nitrate. Water Research , 39, 3982 - 3990. Corwin, A.L., Khiem, H.B., Clayson, E.G., Pham, K.S., Vo, T.T., Vu, T.Y., Cao, T.T., Vaughn, D., Merven, J., Richie , T.L., Putri, M.P., He, J., Graham, R., Wignall, F.S., and Hyams, K.C. (1996). A Waterborne Outbreak of Hepatitis E Virus Transmission in Southwestern Vietnam. The American Journal of Tropical Medicine and Hygiene, 54, 559. Costan - Longares, A., Moce - Llivi an, L., Avellon, A., Jofre, J., Lucena, F. (2008). Occurrence and Distribution of Culture Enteroviruses in Wastewater and Surface Waters of North - Eastern Spain. Journal of Applied Microbiol ogy, 105: 1945 - 1955. Craun, G. F., Brunkard, J. M., Yoder, J. S., R oberts, V. A., Carpenter, J., Wade, T., ... & Roy, S. L. (2010). Causes of outbreaks associated with drinking water in the United States from 1971 to 2006. Clinical Microbiology R eviews , 23 (3), 507 - 528. Crites, R., Tchobanoglous, G. (1998). Small and decen tralized wastewater management systems. The McGraw - Hill Companies. New York, New York. Cruz, J.R., Caceres, P., Cano, F., Flores, J., Bartlett, A., Torun, B. (1990). Adenovirus types 40 and 41 and Rotaviruses Associated with Diarrhea in Children fro Guatem ala. Journal of Clinical Microbiology , 28: 1780 - 1784. Cuthbert, J. A. (2001) Hepatitis A: Old and New. Clinical Microbiology Reviews , 14(1):38. DOI: 10.1128/CMR.14.1.38 - 58.2001. .J. (1979). Pharygoconjunctival Fever Caused by Adenovirus Type 4: Report of a Swimming Pool - Related Outbreak with Recovery of Virus from Pool Water. Journal of Infectious Diseases , 140: 42 - 47. Da Silva, A. K., Le Saux, J - C., Parnaudeau, S., Pommepuy, M., Elimelech, M., Le Guyader, F. S. (2007). Evaluation of removal of noroviruses during wastewater treatment, using real - time reverse transcription - PCR: different behaviors of genogroups I and II. Applied and Environmental Microbiology , 73 (24), 7891 - 7897. Dav is, J. V., & Witt, E. C. (1998). Microbiological quality of public - water supplies in the Ozark plateaus aquifer system, Missouri . US Department of the Interior, US Geological Survey. 45 De Flora, S., De Renzi, G.P., Badolati, G., (1975). Detection of animal v iruses in coastal sea water and sediments. Appl ied Microbiol ogy , 30, 472 - 475. De Paula, V. S., Diniz - Mendes, L., Villar, L. M., Luz, S. B., Jesus, M. S., da Silva, N. M. V. S., Gaspar. A. M. C. (2007). Hepatitis A virus in environmental water samples from the Amazon basin. Water Res earch , 41:1169 - 1176. Dimmock, N., Easton, A., Leppard, K. (2001). Introduction to M odern V irology . 5 th edition. Wiley - Blackwell. Divizia, M., Ruscio, V., Degener, A. M., Pana, A. (1998). Hepatitis A virus detection in wastewater by PCR and hybridization. New Microbiol ogy , 21:161 167. Dong, C., Meng, J., Dai, X., Liang, J. H., Feagins, A. R., Meng, X. J., ... & Teo, C. G. (2011). Restricted enzooticity of hepatitis E virus genotypes 1 to 4 in the United States. Journal of C linical M icrobiology , 49 (12), 4164 - 4172. Donovan, E., Unice, K., Roberts, J. D., Harris, M., & Finley, B. (2008). Risk of gastrointestinal disease associated with exposure to pathogens in the water of the Lower Passaic River. Applied and E nvironmental M icrobiology , 74 (4), 994 - 1003. Dowd, S. E., Pillai, S. D., Wang, S., Corapcioglu, M. Y. (1998). Delineating the specific influence of virus isoelectric point and size on virus adsorption and transport through sandy soils. Applied and Environmental Microbiology , 64 (2), 405 - 410. Dowd, S. E., Gerba, C. P., Pepper, I. L., Pillai, S. D. (2000). Bioaerosol transport modeling and risk assessment in relation to biosolid placement. Journal of Environmental Quality , 29 (1), 343 - 348. Drewry, W. A., Eliassen, R. (1968). Virus movem ent in groundwater. Journal (Water Pollution Control Federation) , 257 - 271. Dziuban, E. J., Liang, J. L., Craun, G. F., Hill, V., Yu, P. A., Painter, J., Moore, M. R., Calderon, R. L., Roy, S. L., Beach, M. J. (2006). Surveillance for waterborne disease and outbreaks associated with recreational water -- United States, 2003 - 2004 . Centers for Disease Control and Prevention (CDC), US Department of Health and Human Services. Eisenberg, J. N., Hubbard, A., Wade, T. J., Sylvester, M. D., LeChevallier, M. W., Levy, D. A., Colford Jr, J. M. (2006). Inferences drawn from a risk assessment compared directly with a randomized trial of a home drinking water intervention. Environmental H ealth P erspectives , 114 (8), 1199. Eisenberg, J. N., Moore, K., Soller, J. A., Eisenberg , D., Colford Jr, J. M. (2008). Microbial risk assessment framework for exposure to amended sludge projects. Environmental H ealth P erspectives , 116 (6), 727. Eischeid, A. C., Meyer, J. N., Linden, K. G. (2009). UV disinfection of adenoviruses: molecular ind ications of DNA damage efficiency. Applied and E nvironmental M icrobiology , 75(1), 23 - 28. Enriquez, C. E., Hurst, C. J., & Gerba, C. P. (1995). Survival of the enteric adenoviruses 40 and 41 in tap, sea, and waste water. Water Research , 29 (11), 2548 - 2553. 46 F arkas, T., Sestak, K., Wei, C., & Jiang, X. (2008). Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae. Journal of V irology , 82 (11), 5408 - 5416. Farrah, S. R., Shah, D. O., Ingram, L. O. (1981). Effects of chaotropic and antichaotropic agents on elution of poliovirus adsorbed on membrane filters. Proceedings of the National Academy of Sciences , 78 (2), 1229 - 1232. Favorov, M. O., Fields, H. A., Purdy, M. A., Yashina, T. L., Aleksandrov, A. G., Alter, M. J., Yarasheva, D. M., Bradley, D. W., Margolis, H. S. (1992). Serologic identification of hepatitis E virus infections in epidemic and endemic settings. Journal of M edical V irology , 36 (4), 246 - 250. Favorov, M. O., Margolis, H. S., & Raoult, D. (1999). Hepatitis E virus inf ection: an enterically transmitted cause of hepatitis. Emerging I nfections , 1 - 16. Fenner, F., White, D. O. (1976). Medical Vrology, 2 nd edition. AcademicPress, Inc. , NewYork. Ferguson, C. M., Coote, B. G., Ashbolt, N. J., & Stevenson, I. M. (1996). Relatio nships between indicators, pathogens and water quality in an estuarine system. Water Research , 30 (9), 2045 - 2054. Fiksdal, L., Leiknes, T. (2006). The effect of coagulation with MF/UF membrane filtration for the removal of virus in drinking water. Journal o f Membrane Science 279, 364 - 371. Fong, T - T., Griffin, D. W., Lipp, E. K. (2005). Molecular assays for targeting human and bovine enteric viruses in coastal waters and their application for library - independent source tracking. Applied and Environmental Micr obiology , 71(4):2070. Fong, T. T., Mansfield, L. S., Wilson, D. L., Schwab, D. J., Molloy, S. L., & Rose, J. B. (2007). Massive microbiological groundwater contamination associated with a waterborne outbreak in Lake Erie, South Bass Island, Ohio. Environmen tal health perspectives , 856 - 864 . Fout, G. S., Martinson, B. C., Moyer, M. W. N., Dahling, D. R. (2003). A Multiplex Reverse Transcription - PCR Method for Detection of Human Enteric Viruses in Groundwater. Applied Envirnmental Microbiol ogy, 69(6): 3158 - 3164 . Foy, H. M. (1997). Adenoviruses, p. 119 - 138. A.S. Evans and R.A. Karlow (ed.) Viral I nfection in H umans: E pidemiology and C ontrol, 4 th ed. Plenum Press, New York, NY. Foy, H. M., Cooney M. K., Hatlen, J. B. (1968). Adenovirus Type 3 Epidemic Associated w ith Intermittent Chlorination of a Swimming Pool. Archives of Environmental Health , 17:795 - 802. Fuhs, G. W., Chen, M., Sturman, L. S., Moore, R. S. (1985). Virus adsorption to mineral surfaces is reduced by microbial overgrowth and organic coatings. Microb ial E cology , 11 (1), 25 - 39. Gannon, V. P. J., Bolin, C., Moe, C. L. (2004). Waterborne Zoonoses: Emerging Pathogens and Emerging Patterns of Infection. Waterborne Zoonoses: Identification, Causes and Control , Edited by J. A. Cotruvo, A. Dufour, G. Rees, J. Bartram, R. Carr, D. O. Cliver, G. F. Craun, R. Fayer, V. P. J. Gannon, WHO. 47 Gerba, C. P. (1981). Virus survival in wastewater treatment (Vol. 39). Pergamon Press, New York. Gerba, C. P. (2007). Virus occurrence and survival in the environmental waters. Pe rspectives in Medical Virology , 17 , 91 - 108. Gerba, C. P., Bitton, G. (1984). Microorganisms as groundwater tracers . New York, John Wiley & Sons, 225 - 233. Gerba, C. P., Lance, J. C. (1978). Poliovirus removal from primary and secondary sewage effluent by so il filtration. Applied and E nvironmental M icrobiology , 36 (2), 247 - 251. Gerba, C. P., Stagg, C. H., Abadie, M. G. (1978). Characterization of sewage solid - associated viruses and behavior in natural waters. Water R esearch , 12 (10), 805 - 812. Gerba, C. P., Meln ick, J. L., Wallis, C. (1975). Fate of wastewater bacteria and viruses in soil. Journal of the I rrigation and D rainage D ivision , 101 (3), 157 - 174. Gerba, C. P., Pepper, I. L., Whitehead 3rd, L. F. (2002a). A risk assessment of emerging pathogens of concern in the land application of biosolids. Water science and technology: a journal of the International Association on Water Pollution Research , 46 (10), 225. Gerba, C. P., Gramos, D. M., Nwachuku, N. (2002b). Comparative inactivation of enteroviruses and adenov irus 2 by UV light. Applied and E nvironmental M icrobiology , 68(10), 5167 - 5169. Gerba, C. P., Rose, J. B. (1990). Viruses in source and drinking water. IN: Drinking Water Microbiology: Progress and Recent Developments. Springer - Verlag New York, Inc., New Yo rk. 1990. p 380 - 396, 5 tab, 64 ref.. Gitis, V., Adin, A., Nasser, A., Gun, J., Lev, O. (2002). Fluorescent dye labeled bacteriophages a new tracer for the investigation of viral transport in porous media: 1. Introduction and characterization. Water R esearc h , 36 (17), 4227 - 4234. Gleick, P. H. (2002). Dirty Water: Estimated Deaths from Water - Related Diseases 2000 - 2020. Pacific Institute Research Report. Pacific Institute for Studies in Development, Environment, and Security . Goodridge, L., Goodridge, C., Wu, J.Q., Griffiths, M. and Pawliszyn, J. (2004). Isoelectric point determination of norovirus virus - like particles by capillary Isoelectric focusing with whole column imaging detection. Analytical Chemistry , 76, 48 52. Gordon, D.M. (2001). Geographical struct ure and host specificity in bacteria and the implications for tracing the source of coliform contamination. Microbiology , 147: 1079 1085. Gordon, C., Toze, S. (2003). Influence of groundwater characteristics on the survival of enteric viruses. Journal of A pplied M icrobiology , 95 (3), 536 - 544. Gourmelon, M., Caprais, M. P., Le Mennec, C., Mieszkin, S., Ponthoreau, C., & Gendronneau, M. (2010). Application of library - independent microbial source tracking methods for identifying the sources of faecal contaminat ion in coastal areas. Water Science a nd Technology , 61 (6), 1401 - 1409. 48 Grce, M., Pavelic, K. (2004). Antiviral properties of clinoptilolite. Microporous and Mesoporous Materials 79 (2005) 165 169. Gujer, W., Henze, M., Mino, T., van Loodsrecht, M., (1999). Activated sludge model no. 3. Water Sci ence Technol ogy , 39(1), 183 - 193. Guo, H., Chu, X., Hu, J. (2010). Effect of host cells on low - and medium - pressure UV inactivation of adenoviruses. Applied and E nvironmental M icrobiology , 76(21), 7068 - 7075. Hagedorn, C., Blanch, A. R., Harwood, V. J. (Eds.). (2011). Microbial source tracking: methods, applications, and case studies . Springer. Haramoto, E., Katayama, H., Oguma, K., Ohgaki, S. (2007). Quantitative analysis of human enteric adenoviruses in aquatic enviro nments. Journal of A pplied M icrobiology , 103: 2153 - 2159. Harwood, V. J., Brownell, M., Wang, S., Lepo, J., Ellender, R. D., Ajidahun, A., Hellein, K. N., Kennedy, E., Ye, X., Flood, C. (2009). Validation and field testing of library - independent microbial s ource tracking methods in the Gulf of Mexico. water research , 43 (19), 4812 - 4819. Haas, C. N. (1983). Effect of effluent disinfection on risks of viral disease transmission via recreational water exposure. Journal ( Water Pollution Control Federation ), 1111 - 1 116. Haas, C.N., Rose, J.B. and Gerba, C.P. (1999). Quantitative Microbial Risk Assessment . New York: John Wiley and Sons, Inc. Havelaar, A. H., Van Olphen, M., & Drost, Y. C. (1993). F - specific RNA bacteriophages are adequate model organisms for enteric v iruses in fresh water. Applied and E nvironmental M icrobiology , 59 (9), 2956 - 2962. He, J. W., Jiang, S. (2005). Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real - Time PCR. Applied and Environmental Microbiology , 71:2250 - 22 55. Hejkal, T. W., Keswick, B., LaBelle, R. L., Gerba, C. P., Sanchez, Y., Dreesman, G., Hafkin, B., Melnick, J. L. (1982) . Viruses in a community water supply associated with an outbreak of gastroenteritis and infectious hepatitis. Journal (American Water Works Association) , 318 - 321. Herath, G., Yamamoto, K., Urase, T. (1999). Removal of viruses by microfiltration membranes at different solution environments. Water S cience and T echnology , 40(4), 331 - 338. Herbold, K., Flehmig, B., Botzenhart, K., (1989). Co mparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms. Applied and Environmental Microbiology , 55(11), 2949 - 2953. Hewitt, J., Leonard, M., Greening, G. E., Lewis, G. D. (2011). Influence of wastewater treatment process and the population size on human virus profiles in wastewater. Water Research , 45: 6267 - 6267. 49 Hirani, Z. M., DeCarolis, J. F., Adham, S. S., & Jacangelo, J. G. (2010). Peak flux performance and microbial removal by selected membrane bior eactor systems. Water R esearch , 44 (8), 2431 - 2440. Hlavsa, M. C., Roberts, V. A., Anderson, A. R., Hill, V. R., Kahler, A. M., Orr, M., Garrison, L. E., Hicks, L. A., Newton, A., Hilborn, E. D., Wade, T. J., Beach, M. J., Yoder, J. S. (2011). Surveillance f or Waterborne Disease Outbreaks and Other Health Events Associated with Recreational Water -- United States, 2007 - 2008. US Department of Health and Human Services, Centers for Disease Control and Prevention. Hopkins, R. S., Shillam, P., Gaspard, B., Eisnach, L., Karlin, R. J. (1985) . Waterborne disease in Colorado: three years' surveillance and 18 outbreaks. American J ournal of P ublic H ealth , 75 (3), 254 - 257. Horwitz, M. S., (1996). Adenoviruses. Fields Virology, 3 rd Edition, Fields B.N, Knipe D.M., Howley P.M . (Editors), Lippincott - Raven Publishers, Philadelphia PA. Hu, J. Y., Ong, S. L., Song, L. F., Feng, Y. Y., Liu, W. T., Tan, T. W., Lee, L. Y., Ng, W. J. (2003). Removal of MS2 bacteriophage using membrane technologies. Water Science and Technology , 47 (12) , 163 - 168. Hubalek, Z. (2003). Emerging Human Infectious Diseases: Anthroponoses, Zoonoses, and Sapronoses. Emerging Infectious Diseases . 9(3): 403 - 404. Hughes, J. M., Wilson, M. E., Teshale, E. H., Hu, D. J., Holmberg, S. D. (2010). The two faces of hepat itis E virus. Clinical I nfectious D iseases , 51 (3), 328 - 334. Hundesa, A., de Motes, C. M., Bofill - Mas, S., Albinana - Gimenez, N., & Girones, R. (2006). Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal c ontamination in the environment. Applied and E nvironmental M icrobiology , 72(12), 7886 - 7893. Hundesa, A., Maluquer de Motes, C., Albinana - Gimenez, N., Rodriguez - Manzano, J., Bofill - Mas, S., Sunen, E., & Rosina Girones, R. (2009). Development of a qPCR assa y for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment. Journal of V irological M ethods , 158(1), 130 - 135. Hundesa, A., Bofill - Mas, S., Maluquer de Motes, C., Rodriguez - Manzano, J., Bach, A., Casas, M., & Girones, R. (2010). Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source - tracking tool. Journal of V irological M ethods , 163(2), 385 - 389. Hurst, C. J., McClellan, K. A., Benton, W. H. (1988). Compa rison of Cytopathogenicity, Immunofluerescence and in situ DNA Hybridization as Methods for the Detection of Adenoviruses. Water Research , 22:1547 - 1552 Hurst, C. J., Benton, W. H., McClellan, K. A. (1989). Thermal and water source effects upon the stabilit y of enteroviruses in surface freshwaters. Canadian J ournal of M icrobiology , 35 (4), 474 - 480. ICTV (International Committee on Taxonomy of Viruses), (2012). Virology Division - IUMS. http://www.ictvonline.org/ 50 Irvin g, L. G., Smith, F. A. (1981). One year survey of enteroviruses, adenoviruses, and reoviruses isolated from effluent at an activated - sludge purification plant. Applied and Environmental Microbiology , 41: 51 - 59. Jamieson, R. C., Gordon, R. J., Sharples, K. E., Stratton, G. W., Madani, A. (2002). Movement and persistence of fecal bacteria in agricultural soils and subsurface drainage water: A review. Canadian Biosystems Engineering , 44 (1), 1 - 9. Jansons, J., Edmonds, L. W., Speight, B., Bucens, M. R. (1989). S urvival of viruses in groundwater. Water Research , 23 (3), 301 - 306. Jiang, S. C., Chi, W., He, J - W. (2007). Seasonal Detection of Human Viruses and Coliphage in Newport Bay, California. Applied and Environmental Microbiology , 73:6468 - 6474. Jin, L., Dai, J. , Wang, L., Wei, Z., Huang, Q., Zhang, Z. (1997). Determination and estimation of the sorption of benzaldehydes on soil. Chemosphere , 35 (11), 2707 - 2712. Jin, Y., Flury, M. (2002). Fate and transport of viruses in porous media. Advances in Agronomy , 77 , 39 - 102. Katayama, H., Haramoto, E., Oguma, K., Yamashita, H., Tajima, A., Narajima, H., Ohgaki, S. (2008). One - year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan. Water R esearch , 4 2: 1441 - 1448. Katz, M., Plotkin, S.A. (1967). Minimal Infective Dose of Attenuated Polio Virus for Man. American Journal of Public Health , 37:1837. Katzenelson, E., Koerner, G., Biedermann, N., Peleg, M., Shuval, H.I. (1979). Measurment of the inactivation kinetics of poliovirus by ozone in a fast - flow mixer. Applied and Environmental Microbiology , 37(4): 715 - 718. Keswick, B. H., Gerba, C. P. (1980). Viruses in groundwater. Environmental Science and Technology , 14 (11), 1290 - 1297. Keswick, B. H., Gerba, C. P ., DuPont, H. L., Rose, J. B. (1984). Detection of enteric viruses in treated drinking water . Applied and Environmental Microbiology , 47: 1290 - 1294. Kinoshita, T., Bales, R. C., Maguire, K. M., Gerba, C. P. (1993). Effect of pH on bacteriophage transport t hrough sandy soils. Journal of Contaminant Hydrology , 14 (1), 55 - 70. Kishino, H., Ishida, H., Iwabu, H., & Nakano, I. (1996). Domestic wastewater reuse using a submerged membrane bioreactor. Desalination , 106 (1), 115 - 119. Kitajima, M., Haramoto, E., Phanuwa n, C., Katayama, H., Ohgaki, S. (2009). Detection of genegroup IV norovirus in wastewater and river water in Japan. The society for applied microbiology, Letters in A pplied M icrobiology , 49: 655 - 658. Ko, G., Cromeans, T. L., Sobsey, M. D. (2005). UV inacti vation of adenovirus type 41 measured by cell culture mRNA RT - PCR. Water R esearch , 39: 3643 - 3649. Korsman, S. N., Van Zyl, G., Preiser, W., Nutt, L., Andersson, M. I. (2012). Virology: An 51 Illustrated Colour Text . Churchill Livingstone Elsevier. Krajden, M. , Brown, M., Petrasek, A., Middleton, P. (1990). Clinical Features of Adenovirus Enteritis: A Review of 127 Cases. The Pediatric Infectious Disease Journal , 9(9): 636 - 641. Kramer, M. H., Herwaldt, B. L., Craun, G. F., Calderon, R. L., Juranek, D. D. (1996) . Surveillance for Waterborne - disease Outbreaks: United States, 1993 - 1994 . US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention (CDC). Krauth, K. H., & Staab, K. F. (1993). Pressurized bioreactor wit h membrane filtration for wastewater treatment. Water Research , 27 (3), 405 - 411. Kukkula, M., Arstila, P, Klossner, M.L., Maunula, L., Bonsdorff, C.H.V, Jaatinen, P. (1997) Waterborne outbreak of viral gastroenteritis. Scandinavian Journal of Infectious Dis eases , 29: 415 - 418. Kumar, A., Wong, K., & Xagoraraki, I. (2012). Effect of Detection Methods on Risk Estimates of Exposure to Biosolids Associated Human Enteric Viruses. Risk Analysis , 32 (5), 916 - 929. Kuo, D.H. - W., Simmons F.J., Blair, S., Hart, E., Rose, J.B., Xagoraraki, I. (2010). Assessment of human adenovirus removal in a full - scale membrane bioreactor treating municipal wastewater. Water R esearch , 44: 1520 - 1530. Kwo, P. Y., Schlauder, G. G., Carpenter, H. A., Murphy, P. J., Rosenblatt, J. E., Dawson, G. J., Mast, E. E., Krawczynski, K., Balan, V. (1997, December). Acute hepatitis E by a new isolate acquired in the United States. In Mayo Clinic Proceedings , 72 ( 12 ), 1133 - 1136 . Elsevier. Lapen, D. R. et al., (2008). Effect of liquid municipal biosolid ap plication method on tile and ground water quality. Journal of E nvironmental Q uality , 37 (3), 925 - 936. Leclerc, H., Edberg, S., Pierzo, V., & Delattre, J. M. (2000). Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. Jou rnal of A pplied M icrobiology , 88 (1), 5 - 21. Lee, S. H., Levy, D. A., Craun, G. F., Beach, M. J., Calderon, R. L. (2002). Surveillance for waterborne - disease outbreaks - United States, 1999 - 2000. MORBIDITY AND MORTALITY WEEKLY REPORT CDC SURVEILLANCE SUMMARI ES , 51 (8). Lee, J. E., Lim, M. Y., Kim, S. Y., Lee, S., Lee, H., Oh, H. M., ... & Ko, G. (2009). Molecular characterization of bacteriophages for microbial source tracking in Korea. Applied and E nvironmental M icrobiology , 75 (22), 7107 - 7114. Lepow, M.L.; W arren, R.J.; Ingram, V.G. (1962). Sabin Type I Oral Poliomyelitis Vaccine: Effect of Dose Upon Response of New borne Infants. American Journal of Diseases of Children , 104:67. Levine, W. C., Stephenson, W. T., Craun, G. F. (1990). Waterborne disease outbre aks, 1986 1988. Mmwr Cdc Surveill Summ , 39 (1), 1 - 13. 52 Levy, D. A., Bens, M. S., Craun, G. F., Calderon, R. L., Herwaldt, B. L. (1998). Surveillance for waterborne - disease outbreaks -- United States, 1995 - 1996. MMWR. CDC surveillance summaries: Morbidity and m ortality weekly report. CDC surveillance summaries/Centers for Disease Control , 47 (5), 1. Liang, J. L., Dziuban, E. J., Craun, G. F., Hill, V., Moore, M. R., Gelting, R. J., Calderon, R. L., Beach, M. J., Roy, S. L. (2006). Surveillance for waterborne dise ase and outbreaks associated with drinking water and water not intended for drinking --- United States, 2003 -- 2004. Surveillance Summaries . Lieberman, R. J., Shadix, L. C., Newport, B. S., Crout, S. R., Buescher, S. E., Safferman, R. S., Stetler, R. E., Lye, D., Fout, G. S., Dahling, D. R. (1995). Source water microbial quality of some vulnerable public groundwater supplies. Proc. WQTC, AWWA , Denver Colorado Linden, K. G., Thurston, J., Schaefer, R., Malley, J. P. (2007). Enhanced UV inactivation of adenoviru ses under polychromatic UV lamps . Applied and E nvironmental M icrobiology , 73(23), 7571 - 7574. Linden, K.G., Shin, G. - A., Lee, J. - K., Scheible, K., Shen, C., Posy, P. (2009). Demonstrating 4 - log adenovirus inactivation in a medium - pressure UV disinfection re actor. American W ater W orks As sociation J ournal , 101 (4): 90 - 99. Liu, T. M., Chen, H. P., Wang, L. T., Wang, J. R., Luo, T. N., Chen, Y. J., Liu, S. L., Sun, C. K. (2009). Microwave resonant absorption of viruses through dipolar coupling with confined aco ustic vibrations. Applied Physics Letters , 94 (4), 043902 - 043902. Lodder, W.J., and de Roda Husman A.M. (2005). Presence of noroviruses and other enteric viruses in sewage and surface waters in The Netherlands. Applied and E nvironmental M icrobiology , 71: 14 53 - 1461. Lv, W., Zheng, X., Yang, M., Zhang, Y., Liu, Y., & Liu, J. (2006). Virus removal performance and mechanism of a submerged membrane bioreactor. Process Biochemistry , 41 (2), 299 - 304. Madaeni, S. S., Fane, A. G., & Grohmann, G. S. (1995). Virus remov al from water and wastewater using membranes. Journal of Membrane Science , 102 , 65 - 75. Madigan, M. T., Martinko, J. M. (2006). Brock Biology of Microorganisms , 11 th edition. ISBN 0 - 13 - 144329 - 1. Mahoney, F. J., Farley, T. A., Kelso, K. Y., Wilson, S. A., Ho ran, J. M., & McFarland, L. M. (1992). An outbreak of hepatitis A associated with swimming in a public pool. Journal of Infectious Diseases , 165 (4), 613 - 618. Marshall, G. S. (2009). Rotavirus disease and prevention through vaccination. The Pediatric I nfecti ous D isease J ournal , 28 (4), 351 - 364. Martinez, A. A., Castillo, J., Sanchez, M. C., Zaldivar, Y., Mendoza, Y., Tribaldos, M., Acosta, P., Smith, R. E., & Pascale, J. M. (2012). Molecular diagnosis of echovirus 30 as the etiological agent in an outbreak of aseptic meningitis in Panama: May June 2008. The Journal of Infection in Developing Countries , 6 (12), 836 - 841. 53 W. G. (1980). An Outbreak of Adenovirus Type 3 Dise ase at a Private Recreation Center Swimming Pool. American Journal of Epidemiology , 111: 229 - 237. Masago, Y., Katayama, H., Watanabe, T., Haramoto, E., Hashimoto, A., Omura, T., Hirata, T., Ohgaki, S. (2006). Quantitative risk assessment of noroviruses in drinking water based on qualitative data in Japan. Environmental S cience and T echnology , 40 (23), 7428 - 7433. Mawdsley, J. L., Bardgett, R. D., Merry, R. J., Pain, B. F., Theodorou, M. K. (1995). Pathogens in livestock waste, their potential for movement th rough soil and environmental pollution. Applied Soil Ecology , 2 (1), 1 - 15. Mazaheri Nezhad Fard, R., Barton, M. D., Heuzenroeder, M. W. (2010). Novel bacteriophages in Enterococcus spp. Current M icrobiology , 60 (6), 400 - 406. Melnick, J. L., Gerba, C. P., Wal lis, C. (1978). Viruses in water. Bulletin of the World Health Organization , 56(4): 499 - 508. Mena, K. D. (2007). Waterborne Viruses: Assessing the Risks. Human Viruses in Water , Albert Bosch (Editor), Chapter 8, pp. 163 - 175, DOI 10.1016/S0168 - 7069(07)1700 8 - 7. Mena, K. D., Gerba, C. P. (2009). Waterborne adenovirus. Reviews of Environmental C ontamination and T oxicology , 133 - 167. Meslin, F. X. (1997).Global Aspects of Emerging and Potential Zoonoses: a WHO Perspective. Emerging Infectious Diseases 3(2): 233 - 228. Miagostovich, M. P., Ferreira, F. F. M., Guimaraes, F. R., Fumian, T. M., Mendes, L. D., Luz, S. L. B., Silva, L. A., Leite, J. P. G. (2008). Molecular Detection and Characterization of Gastroenteritis Viruses Occuring Naturally in the Stream Waters of Manaus, Central Amazonia, Brazil. Applied Environ mental Microbiol ogy, 74:375 - 382. Minor, P. (1987). Animal virus structure, Chapter 6, Picornaviridae, edited by Nermut, M. V. and Steven, A. C. Perspectives in Medical Virology , Vol. 3. Elsevier Science P ublishers, Biomedical Division. Minor, T. E., Allen, C. I., Tsiatis, A. A., Nelson, D. B., D'Alessio, D. J. (1981). Human infective dose determinations for oral poliovirus type 1 vaccine in infants. Journal of Clinical M icrobiology , 13 (2), 388 - 389. Monpoeh o, S., Maul, A., Bonnin, C., Patria, L., Ranarijaona, S., Billaudel, S., Ferré, V. (2004). Clearance of human - pathogenic viruses from sludge: study of four stabilization processes by real - time reverse transcription - PCR and cell culture. Applied and E nviron mental M icrobiology , 70 (9), 5434 - 5440. Monpoeho, S., Maul, A., Mignotte - Cariergues, B., Schwartzbrod, L., Billaudel, S., Ferre, V. (2001). Best viral elution method available for quantification of enteroviruses in sludge by both cell culture and reverse t ranscript - PCR. American S ociety for M icrobiology , 67(6): 2484 - 2488. Moore, A. C., Herwaldt, B. L., Craun, G. F., Calderon, R. L., Highsmith, A. K., Juranek, D. D. (1993). Surveillance for waterborne disease outbreaks United States, 1991 1992. CDC 54 surveilla nce summaries , 42 , 1 - 22. Moore, R. S., Taylor, D. H., Sturman, L. S., Reddy, M. M., Fuhs, G. W. (1981). Poliovirus adsorption by 34 minerals and soils. Applied and E nvironmental M icrobiology , 42 (6), 963 - 975. Morace, G., Aulicino, F. A., Angelozzi, C., Cost anzo, L., Donadio, F., & Rapicetta, M. (2002). Microbial quality of wastewater: detection of hepatitis A virus by reverse transcriptase polymerase chain reaction. Journal of A pplied M icrobiology , 92 (5), 828 - 836. Muniesa, M., García, A., Miró, E., Mirelis, B., Prats, G., Jofre, J., Navarro, F. (2004). - lactamase genes. Emerging Infectious D iseases , 10 (6), 1134. Munoz, S. J., Bradley, D. W., Martin, P., Krawczynski, K., Purdy, M. A., Weterberg, S. (1992). Hepatitis E virus fou nd in patients with apparent fulminant non - A, non - B hepatitis. Hepatology , 16 (4 pt 2), A76. Murray, J. P. (1980). Physical chemistry of virus adsorption and degradation on inorganic surfaces: its relation to waste water treatment. Murray, J. P., Parks, G. A. (1980). Poliovirus adsorption on oxide surfaces. Particulates in Water , 89 , 97 - 133. Washington, DC: American Chemical Society. Nasser, A. M., Battagelli, D. and Sobsey, M. D. (1992). Isoelectric focusing of hepatitis A virus in sucrose gradients. Israel Journal of Medical Sciences , 28, 73. Nermut, M. V. (1987). Animal virus structure, Chapter 23, Adenoviridae, edited by Nermut, M. V. and Steven, A. C. Perspectives in Medical Virology , Vol. 3. Elsevier Science Publishers, Biomedical Division. Noble, R. T. , Allen, S. M., Blackwood, A. D., Chu, W., Jiang, S. C., Lovelace, G. L., Sobsey, M, D., Stewart, J. R., Wait, D. A. (2003). Use of viral pathogens and indicators to differentiate between human and non - human fecal contamination in a microbial source tracki ng comparison study. Journal of Water and Health . Nordgren, J., Matussek, A., Mattsson, A., Svensson, L., Lindgren, P. E. (2009). Prevalence of norovirus and factor influencing virus concentrations during one year in a full - scale wastewater treatment plant . Water R esearch , 43: 1117 - 1125. NRC (National Research Council) (2002). Biosolids applied to land. Natl. Academy Press, Washington, D. C. Nwachuku, N., Gerba, C. P. (2004). Emerging waterborne pathogens: can we kill them all?. Current O pinion in B iotechno logy , 15 (3), 175 - 180. Nwachuku, N., Gerba, C. P., Oswald, A., & Mashadi, F. D. (2005). Comparative inactivation of adenovirus serotypes by UV light disinfection. Applied and E nvironmental M icrobiology , 71 (9), 5633 - 5636. Oh, B. S., Jang, H. Y., Jung, Y. J., & Kang, J. W. (2007). Microfiltration of MS2 bacteriophage: Effect of ozone on membrane fouling. Journal of Membrane 55 Science , 306 (1), 244 - 252. Otawa, K., Lee, S. H., Yamazoe, A., Onuki, M., Satoh, H., Mino, T. (2007). Abundance, diversity, and dynamics of viruses on microorganisms in activated sludge processes. Microbial E cology , 53 (1), 143 - 152. Ottoson, J., Hansen, A., Björlenius, B., Norder, H., & Stenström, T. A. (2006). Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant. Water Research , 40 (7), 1449 - 1457. Palmer, S., Brown, D., Morgan, D. (2005). Early Qualitative Risk Assessment of the Emerging Zoonotic Potential of Animal Diseases. BMJ. 331:1256 - 1260. Pancorbo, O. C., Scheu erman, P. R., Farrah, S. R., Bitton, G. (1981). Effect of sludge type on poliovirus association with and recovery from sludge solids. Canadian Journal of Microbiology , 27 (3), 279 - 287. Pankhania, M., Stephenson, T., & Semmens, M. J. (1994). Hollow fibre bio reactor for wastewater treatment using bubbleless membrane aeration. Water R esearch , 28 (10), 2233 - 2236. Papapetropoulou, M., Vantarakis, A.C. (1998) Detection of Adenovirus Outbreak at a Municipal Swimming Pool by Nested PCR Amplification, Journal of Infect ion , 36: 101 - 103. Parsley, L. C., Consuegra, E. J., Kakirde, K. S., Land, A. M., Harper, W. F., Liles, M. R. (2010). Identification of diverse antimicrobial resistance determinants carried on bacterial, plasmid, or viral metagenomes from an activated sludg e microbial assemblage. Applied and Environmental Microbiology , 76 (11), 3753 - 3757. Payment, P., Plante, R., Cejka, P. (2001). Removal of indicator bacteria, human enteric viruses, Giardia cysts, and Cryptosporidium oocysts at a large wastewater primary tre atment facility. Canadian J ournal of M icrobiology , 47: 188 - 193. Payment, P., Trudel, M., Plante, R. (1985). Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven water treatment plants. Applie d and E nvironmental M icrobiology , 49: 1418 - 1428. Payment, P., and Franco, E. (1993). Clostridium prefringens and somatic coliophages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts. Applied and E nvironmental M icr obiology , 59: 2418 - 2424. Petrinca, A.R., et al. (2009). Presence and environmental circulation of enteric viruses in three different wastewater treatment plants. Journal of A pplied M icrobiology , 106: 1608 - 1617. Pieper, A. P., Ryan, J. N., Harvey, R. W., Am y, G. L., Illangasekare, T. H., Metge, D. W. (1997). Transport and recovery of bacteriophage PRD1 in a sand and gravel aquifer: Effect of sewage - derived organic matter. Environmental Science and Technology , 31 (4), 1163 - 1170. 56 Powell, T., Brion, G. M., Jagto yen, M., Derbyshire, F. (2000). Investigating the effect of carbon shape on virus adsorption. Environmental S cience and T echnology , 34 (13), 2779 - 2783. Powelson, D. K., Gerba, C. P. (1994). Virus removal from sewage effluents during saturated and unsaturate d flow through soil columns. Water Research , 28 (10), 2175 - 2181. Powelson, D. K. (1990). Virus transport and survival in saturated and unsaturated flow through soil columns. Dissertation, University of Arizona. Powelson, D. K., Simpson, J. R., Gerba, C. P. (1991). Effects of organic matter on virus transport in unsaturated flow. Appl ied Environ mental Microbiol ogy , 57:2192 - 2196. Powelson, D. K., Mills, A. L. (1998). Water saturation and surfactant effects on bacterial transport in sand columns. Soil S cience , 163 (9), 694 - 704. Prado, T., Silva, D. M., Guilayn, W. C., Rose, T. L., Gaspar, A. M. C., Miagostovich, M. P. (2011). Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants. Water R esearch , 45 (3), 1287 - 1297. Prescott, J .F. (2004). Antimicrobial chemotherapy. Veterinary Microbiology ed. Hirsh, D. C., Maclachlan, N.J. and Walker, R.L. pp. 26 43. Ames: Blackwell Publishing. Puig, M., Jofre, J., Lucena, F., Allard, A., Wadell, G., Gir ones, R. (1994). Detection of Adenoviruses and Enteroviruses in Polluted Waters by Nested PCR Amplification. Applied Environmental Microbiology , 60:2963 - 2970. Ravindran, V., Tsai, H. H., Williams, M. D., Pirbazari, M. (2009). Hybrid membrane bioreactor tec hnology for small water treatment utilities: Process evaluation and primordial considerations. Journal of Membrane Science 344, 39 - 54. Regli, S., Rose, J. B., Haas, C. N., Gerba, C. P. (1991). Modeling the risk from Giardia and viruses in drinking water. Journal (American Water Works Association) , 76 - 84. Robertson, J. B., Edberg, S. C. (1997). Natural protection of spring and well drinking water against surface microbial contamination. I. Hydrogeological parameters. Critical R eviews in M icrobiology , 23 (2), 143 - 178. Rose, J.B., Dickson, L.J., Farrah, S.R., Carnahan, R.P. (1996). Removal of pathogenic and indicator microorganisms by a full - scale water reclamation facility. Water R esearch , 30: 2785 - 2797. Rosenberg, E., Bittan Banin, G., Sharon, G., Shon, A., H ershko, G., Levy, I., Ron, E. Z. (2010). The phage - driven microbial loop in petroleum bioremediation. Microbial Biotechnology , 3 (4), 467 - 472. Rotbart, H. A. (2000). Viral Meningitis. Seminars in neurology , 20(3):277 - 292, DOI: 10.1055/s - 2000 - 9427. A., Cook, N. (2004). Survival of human enteric viruses in the environment and food. FEMS M icrobiology R eviews , 28 (4), 441 - 453. 57 Sander, M., Schmieger, H. (2001). Method for host - independent detection of generalized transducing bacteriophages in natural hab itats. Applied and E nvironmental M icrobiology , 67 (4), 1490 - 1493. Schaum, C., Cornel, P., Jardin, N. (2005). Possibility for a phosphorus recovery from sewage sludge ash. In Proceedings Conference on the Management of Residues Emanating from Water and Waste water Treatment, Johannesburgs, South Africa, Aug. 9 - 12, 2005. Schiff, G. M., Stefanovic, G. M., Young, E. C., Sander, D. S., Pennekamp, J. K., Ward, R. L. (1984). Studies of echovirus - 12 in volunteers: determination of minimal infectious dose and the effe ct of previous infection on infectious dose. Journal of infectious diseases , 150 (6), 858 - 866. Schijven, J. F., Hassanizadeh, S. M. (2000). Removal of viruses by soil passage: Overview of modeling, processes, and parameters. Critical Reviews in Environmenta l Science and Technology , 30 (1), 49 - 127. Schoen, M. E., Ashbolt, N. J. (2010). Assessing pathogen risk to swimmers at non - sewage impacted recreational beaches. Environmental S cience & T echnology , 44 (7), 2286 - 2291. Simmons F. J., Kuo, D. H. - W., Xagoraraki, I. (2011). Removal of human enteric viruses by a full - scale membrane bioreactor during municipal wastewater processing. Water R esearch , 45: 2739 - 2750. Simmons, F. J., and Xagoraraki, I. (2011). Release of infectious human enteric viruses by full - scale wast ewater utilities. Water R esearch , 45: 3590 - 3598. Simonet, J., Gantzer, C. (2006). Inactivation of poliovirus 1 and F - specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers. Applied and E nvironmental M icrobiology , 72(12), 7 671 - 7677. Siqueira, A. A., Santelli, A. C. F. S., Alencar Jr, L. R., Dantas, M. P., Dimech, C. P. N., Carmo, G. M. I., ... & Hatch, D. L. (2010). Outbreak of acute gastroenteritis in young children with death due to rotavirus genotype G9 in Rio Branco, Bra zilian Amazon region, 2005. International Journal of Infectious Diseases , 14 (10), e898 - e903. Shang, C., Wong, H. M., & Chen, G. (2005). Bacteriophage MS - 2 removal by submerged membrane bioreactor. Water R esearch , 39 (17), 4211 - 4219. Shapiro, O.H., Kushmaro, A. (2011). Bacteriophage ecology in environmental biotechnology Processes. Current Opinion in Biotechnology . 22:449 455. Shieh, Y. C., Wong, C. I., Krantz, J. A., Hsu, F. C. (2008). Detection of Naturally Occurring Enteroviruses in Waters Using Direct RT - PCR and Integrated Cell Culture - RT - PCR. J. Virol. Methods. 149:184 - 189. Shin, G. A., Sobsey, M. D. (2008). Inactivation of norovirus by chlorine disinfection of water. Water R esearch , 42(17), 4562 - 4568. Shipitalo, M. J., Gibbs, F. (2000). Potential of eart hworm burrows to transmit injected animal wastes to tile drains. Soil Science Society of America Journal , 64 (6), 2103 - 2109. Slifko T. R., Smith, H. V., Rose, J. B. (2000). Emerging parasite zoonoses associates with 58 water and food. International Journal of Parasitology. 30: 1379 - 1393. Smith, D. C. (2006). Microbial source tracking using F - specific coliphages and quantitative PCR (Doctoral dissertation, University of Rhode Island). Sobsey, M. D., Khatib, L. A., Hill, V. R., Alocilja, E., Pillai, S. (2001). P athogens in Animal Wastes and the Impacts of Waste Management Practices on Their Survival, Transport and Fate. White Paper, Midwest Plan Service , Iowa State University. Sobsey, M. D., Shields, P. A., Hauchman, F. H., Hazard, R. L.,CATON III, L. W. (1986). Survival and transport of hepatitis A virus in soils, groundwater and wastewater. Water S cience and T echnology , 18 (10), 97 - 106. Soller, J. A., Olivieri, A. W., Eisenberg, J. N. S., Sakaji, R., Danielson, R. (2004) Evaluation of microbial risk assessment t echniques and applications. WERF 00 - PUM - 3. Straub, T. M., Pepper, I. L., Gerba, C. P. (1993). Virus survival in sewage sludge amended desert soil. Water Science & Technology , 27 (3 - 4), 421 - 424. Stewart, P. L., Burnett, R. M., Cyrklaff, M., & Fuller, S. D. ( 1991). Image reconstruction reveals the complex molecular organization of adenovirus. Cell , 67 (1), 145 - 154. Stewart Pullaro, J., Daugomah, J. W., Chestnut, D. E., Graves, D. A., Sobsey, M. D., & Scott, G. I. (2006). F+ RNA coliphage typing for microbial so urce tracking in surface waters. Journal of A pplied M icrobiology , 101 (5), 1015 - 1026. Swenson, P. D., Wadell, A., Allard, A., Hierholzer, J. C. (2003). Adenoviruses. Manual of Clinical Microbiology , 8 th Edition, Murray P.R. (Editor), ASM Press, Washington D.C. Tam, L. S., Tang, T. W., Lau, G. N., Sharma, K. R., & Chen, G. H. (2007). A pilot study for wastewater reclamation and reuse with MBR/RO and MF/RO systems. Desalination , 202 (1), 106 - 113. Tani, N., Shimamoto, K., Ichimura, K., Nishii, Y., Tomita, S., & Oda, Y. (1992). Enteric virus levels in river water. Water Research , 26 (1), 45 - 48. Teunis, P. F., Moe, C. L., Liu, P., E Miller, S., Lindesmith, L., Baric, R. S., Le Pendu, J., Calderon, R. L. (2008). Norwalk virus: How infectious is it? Journal of M edica l V irology , 80 (8), 1468 - 1476. Thompson, S. S., Flury, M., Yates, M. V., Jury, W. A. (1998). Role of the air - water - solid interface in bacteriophage sorption experiments. Applied and E nvironmental M icrobiology , 64 (1), 304 - 309. Thompson, S. S., Jackson, J. L. , Suva - Castillo, M., Yanko, W. A., El Jack, Z., Kuo, J., Chen, C - L., Williams, F. P., Schnurr, D. P. (2003). Detection of infectious human adenoviruses in tertiary - treated and ultraviolet - disinfected wastewater. Water E nvironment R esearch , 75(2), 163 - 170. Thurston - Enriquez, J. A., Haas, C. N., Jacangelo, J., Gerba, C. P. (2003a). Chlorine inactivation of adenovirus type 40 and feline calicivirus. Applied and E nvironmental M icrobiology , 69(7), 3979 - 3985. 59 Thurston - Enriquez, J. A., Haas, C. N., Jacangelo, J., Riley, K., Gerba, C. P. (2003b). Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Applied and Environmental Microbiology , 69(1), 577 - 582. Thurston - Enriquez, J. A., Haas, C. N., Jacangelo, J., Gerba, C. P. (2005a). Inactivation of enteric adenovirus and feline calicivirus by ozone. Water R esearch , 39(15), 3650 - 3656. Thurston - Enriquez, J. A., Haas, C. N., Jacangelo, J., Gerba, C. P. (2005b). Inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide. Applied and E n vironmental M icrobiology , 71(6): 3100 - 3105. Trilisky, E. I., Lenhoff, A. M. (2007). Sorption processes in ion - exchange chromatography of viruses. Journal of Chromatography A , 1142 (1), 2 - 12. Tsang, T. H., Denison, E. K., Williams, H. V., Venczel, L. V., Gin sberg, M. M., Vugia, D. J. (2000). Acute hepatitis E infection acquired in California. Clinical Infectious Diseases , 30 (3), 618 - 619. Turner, M., Istre, G. R., Beauchamp, H., Baum, M., & Arnold, S. (1987). Community outbreak of adenovirus type 7a infections associated with a swimming pool. Southern M edical J ournal , 80 (6), 712 - 715. Uhnoo, I., Wadell, G., Svensson, L., Olding - Stenkvist, E., Molby R. (1986). Etiology and Epidemiology of Acute Gastroenteritis in Swedish Children. Journal of Infection , 13: 73 - 89. USEPA, (1999a). Wastewater Technique Fact Sheet: Chlorine disinfection. EPA 832 - F - 99 - 062. USEPA, (1999b). Alternative Disinfectants and Oxidants Guidance Manual . EPA 815 - R - 99 - 014. USEPA, (1999c). Wastewater Technique Fact Sheet: Ozone disinfection. EPA 83 2 - F - 99 - 063. USEPA, (2001a). Manual of methods for virology, Chapter 14. EPA 600/4 - 84/013 (N14). USEPA, (2001b). National Primary Drinking Water Standards, Office of Water. U.S. Environmental Protection Agency, Washington, DC. EPA816 - F - 01 00 7. USEPA, (2003 ). Environmental regulations and technology: Control of Pathogens and vector attraction in sewage sludge. EPA/625/R - 92/013. USEPA, (2006). Prepublication of the Ground Water Rule Federal Register Notice. EPA - HQ - OW - 2002 - 0061; FRL - RIN 2040 - AA97. Vaid, A., Ko pp, C., Johnson, W., & Fane, A. G. (1991). Integrated waste water treatment by coupled bioreactor and membrane system. Desalination , 83 (1), 137 - 143. Van der Roest, H. F., Lanrence, D. P., Van Bentem, A. G. N. (2002). Membrane Bioreactors for Municipal Wast ewater Treatment. IWA Publishing, London. Vega, E. (2006). Attachment and survival of viruses on lettuce: role of physicochemical and biotic factors. Ph. D. Dissertation, Texas A&M University. 60 Viau, E., Peccia, J. (2009). Survey of wastewater indicators an d human pathogen genomes in biosolids produced by class A and class B stabilization treatments. Applied and E nvironmental M icrobiology , 75 (1), 164 - 174. Vidaver, A. K., Koski, R. K., VanEtten, J. L. (1973). Bacteriophage phi 6: a lipid - containing virus of P seudomonas phaseolicola. Journal of Virology , 11, 799 850. Wadell, G. (1984). Molecular Epidemiology of Human Adenoviruses. Current Topics in Microbiology and Immunology , 110: 191 - 220. Ward, R. L., Bernstein, D. I., Young, E. C., Sherwood, J. R., Knowlton, D. R., Schiff, G. M. (1986). Human rotavirus studies in volunteers: determination of infectious dose and serological response to infection. Journal of Infectious Diseases , 154 (5), 871 - 880. WHO, (2004). Water Sanitation and Health. WHO, (2007). Weekly Epid emiology R ecord, 285 - 296. Withey, S., Cartmell, E., Avery, L. M., Stephenson, T. (2005). Bacteriophages; Potential for Application in Wastewater Treatment Processes. Science of the Total Environment , 339(1 - 3): 1 - 18. Wong, M., Kumar, L., Jenkins, T. M., Xag oraraki, I., Phanikumar, M. S., & Rose, J. B. (2009). Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human - specific bacteriological marker. W ater R esearch , 43 (4), 1137 - 1149. Wong, K., Fong, T. T., Bibby, K., & Molina, M. (2012a). Application of enteric viruses for fecal pollution source tracking in environmental waters. Environment I nternational , 45 , 151 - 164. Wong, K., Mukherjee, B., Kahler, A. M., Zepp, R., Molina, M. (2012b). In uence of Inorganic Ions on Aggregation and Adsorption Behaviors of Human Adenovirus. Environmental Science and Technology . dx.doi.org/10.1021/es3028764. Wong, K., Onan, B. M., & Xagoraraki, I. (2010). Quantification of enteric viruses, pathogen indicators , and Salmonella bacteria in class B anaerobically digested biosolids by culture and molecular methods. Applied and E nvironmental M icrobiology , 76 (19), 6441 - 6448. Wong, K., Xagoraraki, I., Wallace, J., Bickert, W., Srinivasan, S., Rose, J. B. (2009b). Remo val of viruses and indicators by anaerobic membrane bioreactor treating animal waste. Journal of E nvironmental Q uality , 38 (4), 1694 - 1699. Wong, K., & Xagoraraki, I. (2011). Evaluating the prevalence and genetic diversity of adenovirus and polyomavirus in bovine waste for microbial source tracking. Applied M icrobiology and B iotechnology , 90 (4), 1521 - 1526. Wu, J., Li, H., Huang, X. (2010). Indigenous somatic coliphage removal from a real municipal wastewater by a submerged membrane bioreactor. Water R esearc h , 44 (6), 1853 - 1862. Wu, Q., Liu, W. T. (2009). Determination of virus abundance, diversity and distribution in a municipal wastewater treatment plant. Water R esearch , 43 (4), 1101 - 1109. 61 Xagoraraki, I., Kuo, D. H., Wong, K., Wong, M., Rose. J. B. (2007). O ccurrence of human adenoviruses at two recreational beaches of the great lakes. Applied Environmental Microbiol ogy , 73:7874 - 7881. Xiao, H., Guan, D., Chen, R., Chen, P., Monagin, C., Li, W ., Su, J., Ma, C., Zhang, W., & Ke, C. (2013). Molecular characteriz ation of echovirus 30 - associated outbreak of aseptic meningitis in Guangdong in 2012. Virology Journal , 10 , 263. Yates, M. V., Gerba, C. P., Kelley, L. M. (1985). Virus persistence in groundwater. Applied and Environmental Microbiology , 49 (4), 778 - 781. Yat es, M. V., Yates, S. R. (1988). Virus survival and transport in ground water. Water Science and Technology , 20 (11 - 12), 301 - 307. Yoder, J. S., Blackburn, B. G., Craun, G. F., Hill, V., Levy, D. A., Chen, N., Lee, S. H., Calderon, R. L., Beach, M. J. (2004). Surveillance for waterborne disease outbreaks associated with recreational water - United States, 2001 - 2002. MMWR , 53 , 1 - 22. Yoder, J., Roberts, V., Craun, G. F., Hill, V., Hicks, L. A., Alexander, N. T., Radke, V., Calderon, R. L., Hlavsa, M. C., Beach, M. J., Roy, S. L. (2008)a. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking - United States, 2005 - 2006 . MMWR Surveill Summ , 57 (9), 39 - 62. Yoder, J. S., Hlavsa, M. C., Craun, G. F., Hill, V., Roberts, V., Yu, P. A., Hicks, L. A., Aldexander, N. T., Roy, S. L., Calderon, R. L., Beach, M. J., (2008)b. Surveillance for waterborne disease and outbreaks associated with recreational water use and other aquatic facility - associated health events -- Unit ed States, 2005 - 2006 . Coordinating Center for Health Information and Service, Centers for Disease Control and Prevention (CDC), US Department of Health and Human Services. Yu, Y. X., Wu, J., & Gao, G. H. (2004). Density - functional theory of spherical elect ric double layer - primitive - model electrolyte solutions. The Journal of chemical physics , 120 , 7223. Zanetti, F., De Luca, G., & Sacchetti, R. (2010). Performance of a full - scale membrane bioreactor system in treating municipal wastewater for reuse purposes. Bioresource technology , 101 (10), 3768 - 3771. Zerda, K. S. (1982). Dissertation, Baylor College of Medicine, Huston, TX. Zerda, K. S., Gerba, C. P. (1984). Agarose isoelectrofocusing of intact virions. Journal of V ir ological M ethods , 9 (1), 1 - 6. Zerda, K. S., Gerba, C. P., Hou, K. C., Goyal, S. M. (1985). Adsorption of viruses to charge - modified silica. Applied and E nvironmental M icrobiology , 49 (1), 91 - 95. Xiang, Z., Wenzhou, L., Min, Y., & Junxin, L. (2005). Evaluati on of virus removal in MBR using coliphages T4. Chinese Science Bulletin , 50 (9), 862 - 867. Zheng, X., Liu, J. (2007). Virus rejection with two model human enteric viruses in membrane bioreactor system. Science in China Series B: Chemistry , 50 (3), 397 - 404. 62 Zhuang, J., Jin, Y. (2003). Virus retention and transport through Al - oxide coated sand columns: effects of ionic strength and composition. Journal of C ontaminant H ydrology , 60 (3), 193 - 209. 63 CHAPTER 2 LITERATURE REVIEW: MEMBRANE BIOREA C TORS FOR WATER REU SE IN THE UNITED STATES Abstract Water scarcity is a global problem, and the production of wastewater is growing correspondingly along with the ever increasing water consumption. Wastewater can be used as an alternative water resource. Technological deve lopments in treating municipal wastewater, such as membrane bioreactors, provide high quality effluents appropriate for water reuse. In this chapter, we review water reuse issues and standards in the U.S., features and challenges of membrane bioreactor sys tems, and status of MBR applications in the U.S. It can be concluded that MBR is a superior wastewater treatment technology comparing to conventional activated sludge systems, and it can fulfill the growing water reuse demand. Keywords: membrane bioreacto r, wastewater reuse, pollutant removal, membrane fouling 64 2. 1. Water Reuse in the United States Generally, the United States is not considered as a country w ith severe water scarcity (IWMI 2000). However, it has been reported that precipitation is not ab le to satisfy the withdrawals of fresh water in many regions across the United States, especially in the areas with fast - growing p opulation (Hightower and Pierce 2008). The value of reclaimed water, as an alternative to fresh water sources, has been recogn ized in many countries. China, Mexico and the U.S. are the top three countries regarding to total volume of reused water, but in China and Mexico, around half of the reused water is untreated wastewater. The U.S. ranks the first for reuse of treated water, and the volume is approximately four time s higher than in S audi Arabia, who takes the second place ( Jiménez and Asano 2008 ). Approximate 9.84 million cubic meters of water is reused/reclaimed per day in the U.S., but that only accounts to 7.4% of the tota l volume of wast ewater generated (Miller et al. 2006 , USEPA 2012 ). The volume of reused water is increasing at an annual rate of 15% in the U.S. (Miller et al. 2006). In the U.S., reclaimed water may serve for many purposes, including urban reuse, industri al reuse, agriculture reuse, environmental reuse, ground water recharge, and potable reuse ( US EPA 2012). Agricultural reuse takes the largest portion of 29% of reclaimed water across the country, while landscape/golf course irrigation and recreational impo undment occupy a total of 25% ( Bryk et al. 2011 ; US EPA 2012). The remaining categories of reuses include commercial & industrial reuse, groundwater recharge, g eothermal/ e nergy p roduction , natural system restoration, discharge to wetlands and w ildlife h abit at . California is the most populous state in the United States, and it has the largest surface and ground water withdrawals. The report of California Recycled Water Policy states that Resour ces Control Board 2013). The history of water reuse in California can be traced back to 1890s. In 2009, the recycled water in California has reached 0.8 km 3 , but it is still only a small portion when 65 comparing to the state annual water use, 53 km 3 ( Water R euse Association 2009a). According to the California State Water Resources Control Board (2012), agricultural irrigation takes the largest portion (37%) of the reclaimed water. The percentages for landscape irrigation and golf course irrigation are 17% and 7%, respectively. Aquifer recharge, as an indirect potable reuse, has been implemented in California since 1960s (Water Reuse Association 2010), and now its share is 12%. National Water Research Institute (2012) proposed the possibility of direct water re use in southern California, but it has not been applied so far. It has been estimated that the annual water reuse could reach 2.5 km 3 by 2020, and 3.7 km 3 by 2030 (California Stat e Water Resources Control Board 2009). Florida is a leading state in water re use, where 49% of treated water is reused (Florida Departm ent of Environmental Protection 2012). The total amount of reused water increased from 0.285 km 3 in 1986 to 1 km 3 in 2012, and the per capita reuse flow is 0.14 million m 3 per day in average (Florid a Departm ent of Environmental Protection 2013). A percentage of 55% of reclaimed water is used in public access areas, such as parks and schools. 10% of reclaimed water is used to irrigate more than 56.9 k m 2 of farmland. Industrial reuse and groundwater re charge take 17% and 13% of reclaimed water, respectively. The rapid growing population has been suggested as the major driving force for the high - level of water reuse (Asano et al. 2007). Economic merits may be another driving force. A total of 74 water re use utilities in Florida claimed that they provided reclaimed water to their customers for free (Florida Departm ent of Environmental Protection 2013). As the public is a major stakeholder involved in the decision - making of water management ( National Acad emy of Sciences 2012), social factors play a key role in water reuse (Bouwer 2000). Water reuse projects may fail due to social resistance, even though the treated water can meet certain standards. For example, several indirect potable water reuse projects in the U.S. were strongly opposed by the public. Also, n made people uncomfortable, and the social acceptance for water reuse was fairly low. Social 66 awareness of water reuse is rising in the U.S. A survey conducted by the Water Reuse Research Foundation indicated that people in cities where water reuse projects had been applied were aware of reclaimed water (Water Reuse Association 2009b). However, the levels of water reuse across the U.S. are quite diverse, and it appears that p ublic trust on agencies and confidence on the ability of technologies in pollutant removal were declining (Bruvold 1998). To conclude, water reuse may still be a controversial topic in the U.S. public. 2. 2. Water Reuse Standards Water reuse generally ref for a 2012). It has been considered as an alternative water source in addition to natural water sources. Water reuse can be classified to direct reuse and indirect reuse. Appl ications of reclaimed water coming out from treatment facilities directly to target fields, such as agricultural or landscape irrigation, is referred as direct reuse. Indirect reuse, on the other hand, is the discharge of treated water to water bodies (e.g . streams, groundwater aquifer) or storage in a reservoir (e.g. impoundment) before reuse (Levine and Asano 2004). Water reuse can also be categorized into direct potable reuse, indirect potable reuse, and non - potable reuse, in terms of drinking water supp ly. Non - potable reuse, like agricultural irrigation, has been widely accepted by scientific communities and the general public, whereas potable reuse is still far to reach a c onsensus (Bouwer 2000; Hartley 2006). In 1989, the World Health Organization (WHO ) published a health guideline for the use of wastewater in agriculture and aquaculture. The water quality standards are mainly focus ed on microbial pathogens (WHO 1989). After 3 years, Food and Agriculture Organization (FAO) released its guideline for was tewater treatment and use in agriculture and recommended standards for pH, fecal coliforms, and trace elements ( Pescod 1992). A lot of countries, to name a few, Germany, Japan, China, and Australia, have establi shed their own 67 standards for water reuse (Li et al. 2009). The latest water reuse guide line in the United States was published by EPA in 2012. Based on different reuse applications, water quality criteria are set, and the key parameters include pH, biochemical oxygen demand (BOD), total organic carbo n (TOC), turbidity or total suspended solids, TSS, fecal coliform, and Cl 2 residual, as shown in Table 2 - 1. Nitrogen and phosphorus are not in cluded in the EPA water reuse criteria, but they are considered as water quality monitoring parameters, of which t he treatment goals in reclaimed water are 1 - 30 mg/L and 1 - 20 mg/L, respectively (Levine and Asano 2004). State and local authorities may have additional and stricter standards, depending on the types of reuses. For example, California includes total nitrog en (10mg/L) for indirect potable reuse. North Carolina requires that the level of both Clostridium and coliphage should not exceed 5/100mL (monthly mean) and 25/100mL (daily maximum) in agricultural reuse water (USEPA 2012). 2. 3. Membrane Bioreactors Tech nology for Water Reuse Membrane bioreactors, a combination of activated sludge process with biomass separation by membrane filtration, have become a state - of - the - art technology for municipal and industrial wastewater treatment. Generally there are two ways of integrating the membrane modules into activated sl udge process (Cornel and Krause 2008): (1) the submerged configuration, in which the membranes are immersed in the mixed liquor, and permeate is pumped mechanically or by gravity flow; and (2) the side - stream configuration, in which the activated sludge is pumped through membrane module and then recycled, in order to maintain a constant sludge concentration. Comparatively, submerged (immersed) MBR systems are more cost effective and less energy consuming than tubular side - stream system s (Judd 2011; Daigger 2003). Three membrane modules are available for MBRs: hollow fiber, flat sheet and tubular, of which hollow fiber and flat sheet are more prevalent 68 (Wachinski 2013) . Compared to traditional activated sl udge reactors, advantages of MBR include smaller footprint and better effluent quality. Additionally, operation of MBR systems is easier since the performance variability is less, and it significantly reduces the overall area of treatment plant (Choi et al . 2002). MBRs have become a particularly attractive treatment choice for water reuse. In fact, the global MBR market is expanding rapidly. Membrane bioreactors have been considered as a feasible and promising tool for water reuse (B ixio et al. 2006; Melin et al. 2006; Li et al. 2009; Zanetti et al. 2011). Atasoy et al. (2007) suggested that MBRs can not only reclaim the grey water, but also support the reuse of black water, which is more difficult to be recycled due to its high contamination level. MBR tec hnology is able to treat industrial wastewater and match the requirements for water reuse (Galil and Levinsky 2007; Marrot et al. 2004) as well. Cicek (2003) suggested that MBR technology is capable to remove agricultural wastes, such as pesticides, nitrat es and endocrine disrupting compounds, and therefore it can be applied for agricultural wastewater water reuse, and MBR is a technological option where ultrafilt ration can be applied. The fast descending cost of MB R facilities (Bolzonella et al. 2010) make it further more competitive. Howell et al. (2004) concluded four incentives that promote MBR applications for waste treatment: (1) MBR plants are more compact; (2) expansion of plant capacity is simple; (3) the effluent quality is high; (4) the value of reusing is widely recognized. 2. 4. Membrane Fouling: Major Challenge of MBR Application When membrane filtration is carried out in activated sludge , biosolids, colloidal species, and macromolecular species will deposit and accumulate on membrane surface and lead to a flux and permeability decline. This process is called membrane fouling, and it has been considered as the major obstacle and challenge of the develo pment and application of MBRs, 69 as it increases the maintenance and oper ational costs (Bouhabila et al. 2001; Cornel and Krause 2008). Ji et al. (2008) published a scanning electron microscope (SEM) photograph of fouled membrane surface, which indicates tha t bio - film consists of two layers: an inner gel layer and an outer cake layer. The gel layer is thin and compact, and it is strongly attached on the membrane surface. Shin and Kang (2002) suggested that the formation of gel layer is caused by membrane pore blocking and biomass colonization. In contrast, cake layer is thick, porous, and highly compressible ( Murase et al. 1995 ) and it has been suggested that the formation of the oc deposition (Hwang and Hsueh 2003). Trans - membrane pressure (TMP) is widely used to indicate the extent of membra ne fouling (e.g., Ognier et al. 2002; Ognier et al. 2004; Lee et al. 2001). Higher TMP generally means sever e membrane fouling. Membrane fouling could be reversible or irreversible. Reversible fouling is defined as fouling on the membrane surface that can be removed by physical washing, while irreversible fouling, on the other hand, refers to internal fouling in to the membrane pores, which can only be removed by chemical clean (Chang et al. 2002). Fouling control is one of the most important issues in MBR operation. Mixed liquor suspended solids (MLSS) can largely affect the membrane filtration performance (Lee et al. 2001). High MLSS concentrations can accelerate membrane fouling due to large amounts of foulant and rapid deposition of sludge particles on the membrane surface ( Sato and Ishii 1991 ; Han et al. 2005 ), and it has a direct impact upon cake lay er forma tion (Chang and Kim 2005). However, Hong et al. (2002) observed MLSS exhibited very little influence on permeate flux for the range of 3600 - 8400 mg/L, and they suggested fouling was independent of MLSS concentration until a very high value was reached. Add itionally, Li et al. (2008) even reported a negative correlation between MLSS and membrane fouling resistance. Extracellular polymeric substances (EPS) in activated sludge are composed of multiple classes of macromolecules such as carbohydrates, proteins, nucleic acids, phospholipids and 70 other polymeric compounds found at or outside the cell surface and in the intercellular spac e of microbial aggregates (Judd 2008). High concentration of EPS could affect membrane fouling by increasing viscosity of the mixed liquor (Nagaoka et al. 1996), and filamentou s bacteria growth (Meng et al. 2007). The components of soluble microbial products (SMP) include humic and fulvic acids, polysaccharide, proteins, nucleic acid, organic acids, amino acids, antibiotics, steroids, enzymes, structural components of cells, and products of ene rgy metabolism (Rittmann et al. 1987). Carbohydrate component of the SMP were found to be negatively correlated with mem brane permeability (Reid et al. 2006). A positive correlation between food to microorganisms (F/M) ratio and membrane fouling has been found in previous studies. No evidence indicated that F/M ratio had direct impact on membrane fouling, but it could increase the E PS concentrations (Janga et al. 2007), and in turn cause membrane fouling. Additionally, low F/M ratio equals little substrate per unit biomass, which leads to competition among the microorganisms and results in reduction of the net sludge production (Rosenberger et al. 2002). At steady state, low net sludge production l eads to higher s olids r etention t ime ( SRT ) , and less membrane fouling. Positive correlations have been found between the presence of filamentous bacteria and membrane fouling. Choi et al. (2002) observed the membrane fouling was most serious under filament ous sludge bulking conditions, in which, filamentous bacteria were predominant in the sludge floc. Three mechanisms that filamentous bacteria may affect the membrane fouling are proposed: (1) Filamentous bacteria could change the floc mor phology (Li et al. 2008) and lead to irregular shape of bulking sludge (Meng et al. 2006a); (2) The overgrowth of filamentous bacteria in sludge suspension could form a thick and non - porous cake layer and cause sever e membrane fouling (Meng et al. 2006b); (3) Excessive grow th of filamentous bacteria could indirectly cause membrane fouling by significantly increasing the extracellular polymeric substances (EPS) concentration an d sludge viscosity (Meng et al. 2007). Hydraulic r etention t ime (HRT) indicates the average time tha t wastewater stays in 71 activated sludge reactor. It has been suggested that HRT only has an indirect effect on membr ane fouling (Visvanathan et al. 1997; Chang et al. 2002) by affecting other factors, such as MLSS. SRT indicates the average time that suspen ded solids stay in the activated sludge reactor. SRT is suggested as one of the critical factors controlling SMP conce ntration in reactor (Lee et al. 2003). With prolonged SRT, concentrations of suspended solids and volatile suspended solids in the bi oreac tor increase (Huang et al. 2001), and membrane fouling tends to increase due to severer deposition on membrane surface (Han et al. 2005). Nevertheless, similar to HRT, SRT can only indirectly influence membrane fouling (Chang et al. 2002), but the effect o f changes in SRT on fouling potential is more sensitiv e than that of HRT (Jang et al. 2006). Membrane backwash and chemical clean are the two major ways to mitigate membrane fouling in MBR systems. Membrane backwash is a physical process that removes the loosely attached cake layer. Membrane permeate is commonly used for backwash. The backwash duration varies from seconds to minutes. Chemical clean, on the other hand, is a process that can remove most of the fouling substances from the membrane, and recove r the membrane permeability to a large extent. 2. 5. Pollutant Removal in MBR Systems In order to achieve high removal of pollutants, such as nitrogen and phosphate reduction , MBR systems usually consist of multiple stages, as shown in Table 2 - 2. For exam ple, the wastewater treatment plant (WWTP) in Traverse City, MI is equipped with MBR, and it has a total of six stages: one anaerobic stage, one anoxic stage, three aerobic stages, and one aerobic/ membrane stage (Crawford et al. 2006). The operational para meters (MLSS, SRT, recycle ratio, etc.) may be different among stages. Anaerobic treatment is used at the front end of some MBR systems. 72 2. 5.1. Removal of p hysical and c hemical p ollutants in MBRs In conventional activated sludge system, suspended solids a re mainly removed by primary sedimentation. Secondary sedimentation/ clarification is also responsible for the removal of suspended solids, mostly mixed liquor suspended solids. As shown in Table 2 - 2, MBR systems are able to achieve high removal for suspend ed solids and turbidity, due to small membrane pore sizes. It has been suggested that membranes can act as a near - absolute barrier for suspended solids (Wang et al. 2009; Christian et al. 2010). This allows MBRs to be operated at high MLSS levels (Saddoud et al. 2007; Tazi - Pain et al. 2002), that leads to higher removal for pollutants, such as organic substances. The major removal mechanisms of organic matters in conventional activated sludge systems are adsorption and biodegradation. These two mechanisms are also applied in MBR systems (Cirja et al. 2008), and it has been reported that MBRs can remove organic matter more efficiently comparing to conventional activated sludge systems (Gonzalez et al. 2007). Huang et al. (2000) reported high removals of orga nic matter in a submerge MBR. As shown in Table 2 - 2 , full - scale MBRs can achieve high removals (usually > 95%) for organic substances. Chemical precipitation is a traditional method for phosphate removal. This method is reliable, but costs of chemicals and chemical feed systems may be considerable. An consists of an anaerobic stage, where P is released, and an aerobic stage, where P is uptaken (Crocetti et al. 2002; Oehmen e t al. 2005). This process is widely used in wastewater treatment plants with lower costs, but it is less stable than chemical treatment (Oehmen et al. 2007). As discussed above, MBR systems can be operated at high level of MLSS, and this may enhance the bi o - processes, such as nitrification and EBPR. Also, membranes can effectively remove nitrogen and phosphate associated with large particles. Compare d to other pollutants 73 however, removal of nitrogen and phosphate in MBR systems appears to be less stable. Ex tra attention need s to be paid to the removal of nitrogen and phosphate when designing new MBR systems. Conventional methods and technologies may be employed and integrated to the MBRs. For instance, the SymBio ® technology, which promotes the simultaneous nitrification and denitrification (SNdN), is applied in an MBR plant at Delp hos (OH, USA) (OVIVO case study 2011). 2. 5.2. Removal of p athogens in MBRs R emoving microbial pathogens is critical for water reuse safety. The water reuse guidelines set microbia l requirements in terms of fecal coliform. Bacteria removal in full scale MBR systems are summarized in Table 2 - 3. It can be seen that most full - scale MBRs can achieve high removal efficiency for bacteria, and membrane pore size appears to be an important factor. Aidan et al. (2007) reported that an MBR equipped with 0.8 m ceramic membrane could only remove 39% coliform bacteria, while high or complete removal was reached by using membranes with small er por e sizes (Herrera - Robledo et al. 2010; Hirani et al. 2010; Saddoud et al. 2007). Compared to bacterial indicators, i nvestigations for the removal of specific pathogens in MBRs are relatively rare. Tests for some pathogenic bacteria (e.g. Salmonella spp ., Campylobacter spp ., Crytosporidium , etc. ) were applied in several MBR studies, but no concentrations in the influent (raw wastewater) were detected (Winward et al. 2008; Jefferson et al. 2004). It has been reported that more than 100 types of enteric viruses are excreted in human feces and present in cont aminated waters (Melnick et al. 1978; Havelaar et al. 1993). Enter ic viruses pose a considerable threat to human health due to their low infectious dose and long survival in the environment. Table 2 - 4 shows the removal of bacterial viruses (coliphages) 74 and human viruses, such as adenovirues, enteroviruses and noroviruses in full - scale MBRs. Bench - scale MBR with hydrophobic p olyvinylidene fluoride membrane (pore size = 0.22 µ m) could remove 99% of poliovirus, while ultrafiltration could achieve a complete rejection due to smaller pore size (Madaeni et al. 1995). Presence o f biomass, low trans - membrane pressure and stirring could enha nce the removal (Madaeni et al. 1995). MBR systems with higher HRT and lower SRT seemed to be more efficient in removing viruses (Wu et al. 2010). Gallas - Lindemann et al. (2013) reported high r emoval efficiencies for Giardia cyst (99.4%) and Cryptosporidium (94.2%) in a full - scale MBR. Herrera - Robledo et al. (2010 , 2011) reported high removal of helminth eggs in a bench - scale anaerobic MBR with ultrafiltration membranes. By using a pilot - scale a naerobic MBR, Saddoud et al. (2006, 2007b, 2009) ob served complete removal for helmiinth ova and protozoan cysts. Abdel - Shafy (2008) investigated the removal of protozoan cysts, helminthes eggs, nematodes in a pilot submerged MBR, and the results indicated that the MBR was able to reject all of these microorganisms. MBR treatment is usually followed by disinfection. Three traditional methods are available for disinfection, namely, chlorine disinfection, ozone disinfection, and UV disinfection. Chlorine disi nfection is the most commonly used method in conventiona l activated sludge plants. It is cost - effective and well - established, but the residual and forms of chorine could be toxic, and further dech lorination may be required (EPA 1991a). Ozone is more effect ive than chlorine for disinfection, without any residual left in effluent, however, it is very reactive and corrosive, and costs for the met hod could be considerable (EPA 1991b). UV disinfection leaves no residual in the effluent, but high water turbidity may cau se it to be less effective (EPA 1991c ). As described above, high TSS removal can be achieved in MBRs, which makes UV become a feasible and preferable disinfection process in MBR plants. In fact many MBR plants in the United States use UV for disinfe ction, to name a few: Duvall WWTP (WA), Nantucket WWTP (MA), Cauley Creek WWTP (GA). 75 2. 6. Comparison between Conventional Activated Sludge (CAS) S ystem and MBR Similar to conventional treatment, pretreatment to remove large objects and separate solids and grease from wastewater, is required before the raw wastewater enters MBR systems. Typical components of pretreatment include coarse screen, grit, grease trap, fine screen, equalization, and primary sedimentation. Activated sludge is a component in both CA S and MBR, but different microbial community structures have been observed between the two systems in many prev ious studies (Gao et al. 2004; Ouyang and Liu 2009; Li et al. 2004; Silva et al. 2010; Munz et al. 2008). Furthermore, sludge floc size in MBR sy stems is smaller compari ng to CAS systems (Cicek et al. 1999; Holbrook et al. 2005), which implies higher oxygen transfer rate ( Liu et al. 2001 ). It has been widely accepted that in general MBRs have superior and stable performance in pollutant removal com paring to conventional activated sludge. Soriano et al. (2003) obtained higher carbon and nitrogen removal in an MBR system. Munz et al. (2008) attributed more efficient COD removal and nitrification process in MBR to different microbial community composit ions and distributions. Cirja et al. (2008) concluded that sorption and biodegradation were the major mechanisms of organic micropollutants removal in both CAS and MBR. Although no substantial difference was found between these two systems, the potential c apability of MBR for high organic load was suggested. Gonzalez et al. (2007) showed that concentrations of COD, NH + 4 and total suspended solids (TSS) in MBR effluent were consistently lower than CAS, and it was independent from the influent concentrations. Bernhard et al. (2006) suggested MBR provided better removals for non - adsorbing persistent polar pollutants, such as sulfophenylcarboxylates. Holbrook et al. (2005) concluded that accumulation of nondegradable chemical oxygen demand in MBR was responsible for smaller average floc size and higher observed biological yield coefficient comparing to CAS. Wei et al. (2003) reported that worm growth was much faster in CAS reactor, which might affect effluent quality. Pauwels et al. (2006) found that MBR offered 76 similar removal for ammonium nitrogen and ethinylestradiol when treating hospital water, but had better performance in rejecting indicator microorganisms, such as fecal coliforms. Simmons and Xagoraraki (2011) found higher reduction of human adenoviruses a nd enteroviruses in an MBR plant as compared to CAS plants. 2. 7. Applications of MBR with Water Reuse in the U.S. So far, more than 6000 MBR plants have been installed worldwide, and over 600 of them are in the United States (Kafka 2013). Table 2 - 5 shows MBR plants in the U.S. with water reuse applications. Many MBR facilities provide no information in sight of water re use, where it is more likely that water reuse is not applied. As shown in Table 2 - 5, most MBR plants in the United States began their servi ce after 2004. In fact, a lot of these plants served as conventional WWTPs for decades and were upgraded to MBRs in the 21 st Century, For example, the Union Rome WWTP was initially built in 1986, and commissioned as an MBR in 2009 . The maximum capacities o f most MBR plants are below 38,000 m 3 /d ( 10 mgd), but they could reach 95,000 m 3 /d ( 25 mgd). Construction of an MBR typically takes 1 - 3 years, depending on the size. Additionally, the capacities of MBRs are usually expandable. Kubota and GE appear to be the most prevalent membrane suppliers for MBR facilities across the United States, and they are also the major membrane s u ppliers in Europe (Melin et al. 2006). GE is known for its ZeeWeed membranes, which are a type of ultrafiltration hollow fiber membran e, while Kubota generally provides flat - plate microfiltration membranes. Reclaimed water from MBR systems with reuse programs in the U.S. is mostly used for non - potable purposes, among which land irrigation appears to be one of the most common applications (e.g. Upper Sweetwater WWTP, GA; Corona WWTP, CA). Other non - potable reuse applications include industrial reuse (e.g. Redlands WWTP, CA) and fire protection (e.g. 77 Red Hawk Casino WWTP, CA). Groundwater recharge has been considered as a sustainable and ec onomical way of water storage without eco - environmental problems (Bouwer et al., 2000). Some MBR plants in the U.S. have injected their treated water to underground aquifers, as a type of indirect potable reuse (e.g. Shelton WWTP, WA; Upper Wallkill WWTP, NJ). Although direct potable reuse has been proposed by Water Reuse Association (2009a , 2010), no evidence shows that treated water from MBRs is to be applied for such purpose in the United States. It is notable that many MBR WWTPs discharged their effluen t directly to river without any reuse (e.g. Crooked Creek WRF, GA; Nantucket WWTP, MA), even though the water quality meets the standard for water reuse. In addition, the wastewater treatment facilities with water reuse applications may also discharge a po rtion of their reclaimed water, depending on the demands. For example, the demand of reclaimed water for agricultural irrigation may be low in winter, while treated wastewater is produced all year around. 2. 8. Conclusions Due to the ever - growing water de mand and fierce water crisis all over the world, water reuse and water reclamation, as alternatives to natural water resources, are drawing more and more attention. In the United States, the levels of water reuse are low in general, but increasing fast. Ag ricultural irrigation is generally the most common application of reused water across the country ; other reuses include land irrigation, aquifer recharge, commercial & industrial reuse, wetlands and wildlife habitat . The U.S. Environmental Protection Agenc y ( US EPA) has established guidelines and criteria for water reuse in 2012. Membrane bioreactor (MBR) technology has been prove n as an effective method in wastewater treatment , and provides effluent that meets EPA water reuse criteria. In the U.S., a numbe r of conventional WWTPs have been upgraded to MBR plants, and effluent water is being reused . Previous studies have demonstrated that MBRs have smaller footprints, less 78 land occupancy, and higher removal efficiencies of pollutants , especially organic micro pollutants and emerging pathogens in contrast to conventional activated sludge systems. It has been shown that removal of bacterial indicators and pathogenic viruses in MBR systems as compared to CAS. Membrane fouling is considered as the main obstacle in MBRs, and fouling control is one of the key issues in MBR operation. In the U.S., t reated water from MBR plants is more likely to be reused for land irrigation, such as lawns and golf courses. A lthough membrane technology has been studied for decades and MBR facilities are widely installed in the U.S., the reuse level of reclaimed water from MBRs appears to be low. However, great potential of water reuse is expected when social, economical and environmental drivers are activated. 79 APPENDIX 80 T able 2 - 1. Water quality criteria of EPA guideline for water reuse (EPA 2012) Non - potable reuse I Non - potable reuse II Indirect Potable Reuse Reuse Category Urban reuse (restricted), Processed food corps, Non - food corps, Impoundments (restricted), Enviro nmental reuse, Industrial reuse Urban reuse (unrestricted), Impoundments (unrestricted), Food corps Groundwater recharge into potable aquifers, Augmentation of surface water supply reservoirs pH 6.0 9.0 6.0 9.0 6.5 8.5 Organic matter wastewater origin Turbidity or TSS Fecal coliforms mL No detectable fecal coliform /100 mL No detectable fecal coliform /100 mL Cl 2 residual 1 mg/L Cl 2 (min.) 1 mg/L C l 2 (min.) 1 mg/L Cl 2 (min.) 81 Table 2 - 2. Pollutant removal in selected full - scale MBR plants in the U.S. Plant Configuration and MBR Module Pollutant Removal (Influent/Effluent)* Reference Leoni Twp WWTP, MI Anoxic + pre - aeration + membrane tanks Kubota ® immersed flat sheet membrane UV disinfection TSS ~170/2 Kafka 2013 BOD ~170/2 NH 3 - N 23/0.07 TP 5/0.24 The Hamptons WWTP, GA Anoxic + aerobic stages Kubota ® flat sheet membrane Chemical disinfection TSS 200/<2 Enviroquip case study 2012 Turbidity NA/<0.5 BOD 200/<3 TN 40/<10 TP 10/<0.13 (food industry) Anaerobic Kubota systems Kubota ® immersed flat sheet membrane No disinfection unit reported TSS 12,000/<2 Judd 2011 BOD 18,000/16 COD 34,000/200 Traverse City WWTP, MI Anaerobic + anoxic + aerobic stages ZeeWeed ® immersed hollow fiber membrane UV disinfection TSS 248<1 USEPA 2007; Judd 2011 BOD 280/<2 NH 3 - N 27.9/<0.08 TP 6.9/0.7 Cauley Creek WRF, GA Anaerobic + swing zone + 2 aerobic stages + membrane tanks ZeeWeed ® immersed hollow fiber membrane UV disinfection TSS 174/3.2 USEPA 2007; Badran 2004 BOD 182/2 COD 398/12 TKN 33/1.9 NH 3 - N 24.8/0.21 TP 5/0.1 Calls Creek WWTP, GA Anoxic + aerobic + membrane ta nks Siemens/U.S. Filter Systems Orbal ® system UV disinfection TSS 248/1 USEPA 2007; Pellegrin and Hatcher 2008 Turbidity NA/0.3 BOD 145/1 NH 3 - N 14.8/0.21 TP 0.88/0.28 Redlands WWTP, CA Anoxic + aerobic + membrane tanks ZeeWeed ® reinforce d hollow fiber UF membrane Chlorine disinfection TSS 130/<5 General Electric (GE) case study 2011 Turbidity NA/<0.2 BOD 160/<5 TN 24/<10 Santa Paula WWTP, CA TSS 210/<5 Anoxic + aerobic + membrane tanks BOD 320/<5 Carollo Engineers 2006 P URON ® membrane filtration modules TKN 53/<7 UV disinfection TDS 1300/<1000 *Concentrations of some pollutants in the influent are extrapolated based on the concentrations in the effluent and removal efficiencies. Units for turbidity are NTU, and for ot her parameters are mg/L. 82 Table 2 - 3. Bacteria removal in full - scale MBRs MBR Type (membrane pore size) Bacterial Indicators Removal Efficiency Reference Before disinfection After disinfection Submerged hollow fiber MBR (0.035 m) Total coliform NA U p to 4 logs Bassyouni et at. 2006 Submerged hollow sheet (0.2 m) Total viable count 3.6 logs 4.6 logs Guerra 2010 E. coli 4.7 logs Complete removal Total coliform 4.1 logs Complete removal Submerged MBR (0.4 m) E. coli >98% NA Wen et al. 2004 Su bmerged flat sheet MBR (0.4 m) E. coli 6.35 6.68 logs NA De Luca et al. 2013 Enterococci 5.64 5.84 logs NA Parallel - panel submerged MBR (0.45 m) E. coli 1.7 5.7 logs NA Sima et al. 2011 Submerged flat sheet MBR (0.4 m) Total coliforms 6.02 lo gs 6.93 logs Zanetti et al. 2010 Thermo - tolerant coliforms 6.72 logs 7.32 logs Fecal coliforms 6.98 logs Complete removal E. coli 6.77 logs Complete removal Enterococci 5.77 logs Complete removal Microfiltration MBR (<0.4 m) E. coli 5.37 - > 6.85 logs Not enhanced Francy et al. 2012 Enterococci 4.82 - 7.49 logs Not enhanced Fecal coliforms 5.34 - 7.23 logs Removal enhanced by > 0.30 log 83 Table 2 - 4. Virus removal in full - scale MBRs MBR Type (membrane pore size) Virus Removal Efficiency Reference Before disinfection After disinfection Submerged flat sheet MBR (0.4 m) F - specific coliphage 5.82 logs Complete removal Zanetti et al. 2010 Somatic coliphage 4.44 logs 5.98 logs Bacteriophages infecting bacteroides fragilis Complete removal Complete removal Submerged hollow fiber MBR (0.1 m) HAdV 4.1 - 5.6 logs NA Ku o et al. 2010 Submerged MBR (0.4 m) Norovirus I NA 0 - 5.3 logs* da Silva et al. 2007 Norovirus II NA 0 - 5.5 logs* Submerged hollow fiber MBR (0.1 m) HAdV 3.4 - 4.5 logs* Removal enhanced by ~0.8 log* Simmons and Xagoraraki 2011 Enterovirus 2.9 - 4.6 logs* Removal enhanced by ~0.4 log* Submerged hollow fiber MBR (0.1 m) HAdV 4.1 - 6.3 logs NA Simmons et al. 2011 Enterovirus 4.1 - 6.8 logs NA Norovirus (II) 3.5 - 4.8 logs NA Microfiltration MBR (<0.4 m) F - specific coliphage >4.58 - >6 lo gs Not enhanced Francy et al. 2012 Somatic coliphage 2.67 4.04 logs Removal enhanced by >2.18 logs^ Adenovirus 2.38 - >4.86 logs Not enhanced Enerovirus >2.2 4.74 logs Not enhanced Norovirus I >1.51 3.32 logs Not enhanced Culturable vir uses >1.99 - >3.61 logs Not enhanced Flat sheet submerged MBR (0.4 m) Somatic coliphage 4.43 4.44 logs NA Luca et al. 2013 F - specific coliphage 5.81 5.83 logs NA Parallel - panel submerged MBR (0.45 m) Norovirus 0.9 6.8 logs NA Sima et al. 2011 Sapovirus 1.7 4.1 logs NA *Read from graphs. ^Median value 84 T able 2 - 5. Selected MBR wastewater treatment facilities in the U.S. with water reuse* Name Location Commission Year Peak Capacity Membrane manufacturer Water Reuse Carnation WWTP King County, WA 2008 1817 m 3 /d GE Irrigation Brightwater WWTP King County, WA 2011 117,348 m 3 /d ZeeWeed (GE) Irrigation, industrial reuse Cauley Creek WRF Fulton County, GA 2004 18,927 m 3 /d ZeeWeed (GE) Land irrigation, lawn watering, discharge Fowler WRF Forsyth County, GA 2004 9,464 m 3 /d Zenon (GE) Land irrigation Spokane C ounty WRF Spokane County, WA 2011 30,283 m 3 /d GE Industrial, urban irrigation, wetlands restoration, aquifer recharge Yellow River WRF Gwinnett County, GA 2012 69,273 m 3 /d ZeeWeed (GE) Non - potable purpose or direct discharge to river James Creek WRF Fors yth County, GA 2006 3,785 m 3 /d Enviroquip (Kubota) Land irrigation Johns Creek Environmental Campus Fulton County, GA 2009 56,781 m 3 /d Zenon Irrigation, toilet water, fire protection Pooler WWTP Chatham County, GA 2004 9,464 m 3 /d ZeeWeed Irrigation to g olf course Upper Sweetwater WWTP Paulding County, GA Before 2009 3,785 m 3 /d Kubota Irrigation to golf course Yakama Nation Legends Casino WWTP Yakima County, WA 2008 1,363 m 3 /d Enviroquip (Kubota) Lawn irrigation, discharge Shelton WWTP Mason County, WA 2012 15,142 m 3 /d Ovivo ( Kubota) Regional Plan participants, Ground water recharge, Red Hawk Casino WWTP CA 2008 2,650 m 3 /d Kubota Toilet flushing, fire protection, landscaping American Canyon WWTP Napa County, CA 2002 14,195 m 3 /d ZeeWeed Vineyard and golf course irrigation, discharge Corona WWTP Riverside County, CA 2001 3,785 m 3 /d ZeeWeed Landscape irrigation, discharge Marco Island WWTP Collier County, FL 2007 11,356 m 3 /d ZeeWeed Land irrigation Ironhouse Sanitary District WWTP Contra Costa Count y, CA 2011 32,555 m 3 /d ZeeWeed Irrigation, discharge 85 T able 2 - 5. Fallingwater Conservancy WWTP Fayette County, PA 2003 3,331 m 3 /d ZeeWeed Flush water, garden irrigation Redlands WWTP San Bernardino County, CA 2004 24,984 m 3 /d ZeeWeed Indu strial reuse The Hamptons WRF Forsyth County, GA 2003 1041 m 3 /d Kubota Land irrigation Santa Paula WWTP Ventura County, CA 2010 27,255 m 3 /d Koch membrane Irrigation Upper Wallkill WWTP Sussex County, NJ 2010 1,003 m 3 /d Kubota Groundwater discharge *Man y MBR facilities in the United States are not included in this table due to lack of information regarding water reuse MGD = Million Gallons per Day WWTP = Wastewater Treatment Plant WRF = Water Reclamation Facility Discharge = discharge to the environment (rivers, creeks, canals etc.) 86 REFERENCES 87 R EFERENCES Atasoy, E., Murat, S., Baban, A., & Tiris, M. (2007). Membrane bioreactor (MBR) treatment of segregated household wastewater for reuse. CLEAN Soil, Air, Water , 35 (5), 465 - 472. Badran, (200 4). Immersed Membrane Bioreactors. For Water Reuse: Summary of 5 Years Experience. Zenon Environmental Inc. http://sawea.org/pdf/2004/March - 21 - 2004/I mmersedMembraneBioreactorsForWaterReuseSu mmary.pdf Bassyouni, A., Garcia, G., & Mc Dermott, D. (2006). Small Community Treatment Plant Expansion While in Operation Using Advanced Technology. Proceedings of the Water Environment Federation , 2006 (10), 3018 - 3024. Bernhard, M., Müller, J., & Knepper, T. P. (2006). Biodegradation of persistent polar pollutants in wastewater: comparison of an optimised lab - scale membrane bioreactor and activated sludge treatment. Water R esearch , 40 (18), 3419 - 3428. Bixio, D., Tho eye, C., De Koning, J., Joksimovic, D., Savic, D., Wintgens, T., & Melin, T. (2006). Wastewater reuse in Europe. Desalination , 187 (1), 89 - 101. Bolzonella, D., Fatone, F., di Fabio, S., & Cecchi, F. (2010). Application of membrane bioreactor technology for wastewater treatment and reuse in the Mediterranean region: Focusing on removal efficiency of non - conventional pollutants. Journal of Environmental M anagement , 91 (12), 2424 - 2431. , R., & Buisson, H. (2001). Fouling characterisation in membrane bioreactors. Separation and Purification Technology , 22 , 123 - 132. Bouwer, H. (2000). Integrated water management: emerging issues and challenges. Agricultural W ater M anagement , 45 (3), 217 - 228. Bruvold, W. H. (1998). Public opinion on water reuse options. J. Water Pollution Control Federation, 60(1) 45 50. Bryk, J., R. Prasad, T. Lindley, S. Davis, and G. Carpenter. 2011. National Database of Water Reuse Facilities: Su mmary Report. WateReuse Foundation. Alexandria, VA. California State Water Resources Control Board, 2012. Municipal Wastewater Recycle Survey. Water Recycling Funding Program. http://www.waterboards.ca.gov/water_issues/programs/grants_loans/water_recycling/munirec .shtml California State Water Resources Control Board (California SWRCB ). 2009. Recycled Water Policy . http://www.swrcb.ca.gov/water_issues/programs/water_recycling_policy Carollo Engineers, (2006). City of Santa Paula, Water Recycling Facility Project. Technical Memorandum Economic Alternative Assessment. C icek, N. (2003). A review of membrane bioreactors and their potential application in the 88 treatment of agricultural wastewater. Canadian Biosystems Engineering , 45 , 6 - 37. Chang, I. S., Le Clech, P., Jefferson, B., Judd, S. (2002). Membrane fouling in membra ne bioreactors for wastewater treatment. Journal of Environmental Engineering , 128(11), 1018 - 1029. Chang, I. S., Kim, S. N. (2005). Wastewater treatment using membrane filtration effect of biosolids concentration on cake resistance. Process Biochemistry , 4 0 (3 4), 1307 1314. Cieck, N., Franco, J. P., Suidan, M. T., Urbain, V., & Manem, J. (1999). Characterization and comparison of a membrane bioreactor and a conventional activated - sludge system in the treatment of wastewater containing high - molecular - weight compounds. Water Environment Research , 71 (1), 64 - 70. Cirja, M., Ivashechkin, P., Schäffer, A., & Corvini, P. F. (2008). Factors affecting the removal of organic micropollutants from wastewater in conventional treatment plants (CTP) and membrane bioreactor s (MBR). Reviews in Environmental Science and Bio/Technology , 7 (1), 61 - 78. Choi, J. G., Bae, T. H., Kim, J. H., Tak, T. M., & Randall, A. A. (2002). The behavior of membrane fouling initiation on the crossflow membrane bioreactor system. Journal of Membran e Science , 203 (1), 103 - 113. Côté, P., Siverns, S., & Monti, S. (2005). Comparison of membrane - based solutions for water reclamation and desalination. Desalination , 182 (1), 251 - 257. Cornel, P., & Krause, S. (2008). Membrane bioreactors for wastewater treatm ent. Advanced Membrane Technology and Applications , 217 - 238. Crawford, G., Daigger, G., & Erdal, Z. (2006). Enhanced biological phosphorus removal within membrane bioreactors. Proceedings of the Water Environment Federation , 2006 (11), 1856 - 1867. Crocetti, G. R., Banfield, J. F., Keller, J., Bond, P. L., & Blackall, L. L. (2002). Glycogen - accumulating organisms in laboratory - scale and full - scale wastewater treatment processes. Microbiology , 148 (11), 3353 - 3364. Da Silva, A. K., Le Saux, J - C., Parnaudeau, S., Pommepuy, M., Elimelech, M., Le Guyader, F. S. (2007). Evaluation of removal of noroviruses during wastewater treatment, using real - time reverse transcription - PCR: different behaviors of genogroups I and II. Applied and Environmental Microbiology , Vol. 73, No. 24, pp. 7891 7897. Daigger, G. T. (2003). State of the art of membrane bioreactors in North America. In Proceedings of the International Conference Application and Perspectives of MBRs in Wastewater Treatment and Reuse, Cremona, Apr. 28 - 29. De Luca, G ., Sacchetti, R., Leoni, E., & Zanetti, F. (2013). Removal of indicator bacteriophages from municipal wastewater by a full - scale membrane bioreactor and a conventional activated sludge process: Implications to water reuse. Bioresource T echnology , 129 , 526 - 531. Environmental Protection Agency, (1999 a ). Wastewater Technique Fact Sheet: Chlorine Disinfection. EPA 832 - F - 99 - 062. 89 Environmental Protection Agency, (1999 b ). Wastewater Technique Fact Sheet: Ozone Disinfection. EPA 832 - F - 99 - 063. Environmental Protecti on Agency, (1999 c ). Wastewater Technique Fact Sheet: Ultraviolet Disinfection. EPA 832 - F - 99 - 064. Enviroquip case study, (2012). Georgia Development Selects Enviroquip MBR Technology For Expansion of Water Reclamation Facility. http://www.kubota - mbr.com/municipal/The%20Hamptons%20(USA).pdf Florida Department of Environmental Protection, 2012. 2011 Reuse Inventory. Water Reuse Program. http://edocs.dlis.state.fl.us/fldocs/dep/water/reuse/2011.pdf Florida Department of Environmental Protection, 2013. 2012 Reuse Inventory. Water Reuse Program. http://www.dep.state.fl.us/water/reuse/docs/inventory/2012_reuse - report.pdf Francy, D. S., Stelzer, E. A., Bushon, R. N., Brady, A. M., Williston, A. G., Riddell, K. R., ... & Gellner, T. M. (2012). Comparative effectiveness of mem brane bioreactors, conventional secondary treatment, and chlorine and UV disinfection to remove microorganisms from municipal wastewaters. Water R esearch , 46 (13), 4164 - 4178. Galil, N. I., & Levinsky, Y. (2007). Sustainable reclamation and reuse of industri al wastewater including membran e bioreactor technologies: case studies. Desalination , 202 (1), 411 - 417. Gallas - Lindemann, C., Sotiriadou, I., Plutzer, J., & Karanis, P. (2013). Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiology and I nfection , 141 (01), 9 - 21. Gao, M., Yang, M., Li, H., Yang, Q., & Zhang, Y. (2004). Comparison between a submerged membrane bioreactor and a conventional activated sludge syst em on treating ammonia - bearing inorganic wastewater. Journal of B iotechnology , 108 (3), 265 - 269. General Electric case study, (2011). City of Redlands Wastewater Treatment Plant. CS - RDLDS - MUNWW - EN - 1206 - NA GE Logo.doc Gonzalez, S., Petrovic, M., & Barcelo, D. (2007). Removal of a broad range of surfactants from municipal wastewater comparison between membrane bioreactor and conventional activated sludge treatment. Chemosphere , 67 (2), 335 - 343. Guerra, L. (2010). Evaluation of Bassussarry Wastewater Treatment Plant after Upgrading with Membrane Bioreactor Technology. Han, S. S., Bae, T. H., Jang, G. G., & Tak, T. M. (2005). Influence of sludge retention time on membrane fouling and bioactivities in membrane bioreactor system. Process Biochemistry , 40 (7), 2393 - 2400. Hartley, T. W. (2006). Public perception and participation in water reuse. Desalination , 187 (1), 115 - 126. Havelaar, A. H., Van Olphen, M., & Drost, Y. C. (1993). F - specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water. Applied and environmental microbiology , 59 (9), 2956 - 2962. 90 Herrera - Robledo, M., Cid - Leon, D. M., Morgan - Sagastume, J. M., & Noyola, A. (2011). Biofouling in an anaerobic membrane bioreactor treating municipal sewage. Separation and Purification Techn ology , 81 (1), 49 - 55. Herrera - Robledo, M., Morgan - Sagastume, J. M., & Noyola, A. (2010). Biofouling and pollutant removal during long - term operation of an anaerobic membrane bioreactor treating municipal wastewater. Biofouling , 26 (1), 23 - 30. Hightower, M., & Pierce, S. A. (2008). The energy challenge. Nature , 452 (7185), 285 - 286. Hong, S. P., Bae, T. H., Tak, T. M., Hong, S., & Randall, A. (2002). Fouling control in activated sludge submerged hollow fiber membrane bioreactors. Desalination , 143 (3), 219 - 228. Hi rani, Z. M., DeCarolis, J. F., Adham, S. S., & Jacangelo, J. G. (2010). Peak flux performance and microbial removal by selected membrane bioreactor systems. Water R esearch , 44 (8), 2431 - 2440. Holbrook, R. D., Massie, K. A., & Novak, J. T. (2005). A comparis on of membrane bioreactor and conventional - activated - sludge mixed liquor and biosolids characteristics. Water E nvironment R esearch , 323 - 330. Howell, J. A. (2004). Future of membranes and membrane reactors in green technologies and for water reuse. Desalina tion , 162 , 1 - 11. Huang, X., Gui, P., & Qian, Y. (2001). Effect of sludge retention time on microbial behaviour in a submerged membrane bioreactor. Process Biochemistry , 36 (10), 1001 - 1006. Huang, X., Liu, R., & Qian, Y. (2000). Behaviour of soluble microbia l products in a membrane bioreactor. Process Biochemistry , 36 (5), 401 - 406. Hwang, K. J., & Hsueh, C. L. (2003). Dynamic analysis of cake properties in microfiltration of soft colloids. Journal of M embrane S cience , 214 (2), 259 - 273. International Water Manag ement Institute (IWMI), 2000. World Water Demand and Supply, 1990 to 2025: Scenarios and Issues. Research Report, 19. Jang, N., Ren, X., Cho, J., & Kim, I. S. (2006). Steady - state modeling of bio - fouling potentials with respect to the biological kinetics i n the submerged membrane bioreactor (SMBR). Journal of M embrane S cience , 284 (1), 352 - 360. Janga, N., Ren, X., Kim, G., Ahn, C., Cho, J., & Kim, I. S. (2007). Characteristics of soluble microbial products and extracellular polymeric substances in the membra ne bioreactor for water reuse. Desalination , 202 (1), 90 - 98. Jefferson, B., Palmer, A., Jeffrey, P., Stuetz, R., & Judd, S. (2004). Grey water characterisation and its impact on the selection and operation of technologies for urban reuse. Water Science and Technology , 50 (2), 157 - 164. Ji, J., Qiu, J., Wong, F. S., & Li, Y. (2008). Enhancement of filterability in MBR achieved by improvement of supernatant and floc characteristics via filter aids addition. Water R esearch , 42 (14), 3611 - 3622. 91 Jimenez, B., & Asano , T. (2008). Water reclamation and reuse around the world. Water Reuse: an international survey of current practice, issues and needs , (20), 3. Judd, S. (2008). The status of membrane bioreactor technology. Trends in B iotechnology , 26 (2), 109 - 116. Judd, S. and Judd, C. (2011). The MBR Book Principles and Applications of Membrane Bioreactors in Water and Wastewater Treatment, Elsevier. Great Britain . Kafka, S. (2013). Flat Plate MBRs A Viable and Proven Technology. Hamlett Environmental Technologies Co. http://www.mi - wea.org/docs/Flat%20Plate%20MBR%20 - %20Scott%20Kafka%20 - %202 013%20Process%20Seminar.pdf Kuo, D.H. - W., Simmons F.J., Blair, S., Ha rt, E., Rose, J.B., Xagoraraki, I. (2010). Assessment of human adenovirus removal in a full - scale membrane bioreactor treating municipal wastewater. Water R esearch , 44: 1520 - 1530. Lee, J., Ahn, W. Y., & Lee, C. H. (2001). Comparison of the filtration chara cteristics between attached and suspended growth microorganisms in submerged membrane bioreactor. Water Research , 35 (10), 2435 - 2445. Lee, W., Kang, S., & Shin, H. (2003). Sludge characteristics and their contribution to microfiltration in submerged membran e bioreactors. Journal of Membrane Science , 216 (1), 217 - 227. Levine, A. D., & Asano, T. (2004). Peer reviewed: Recovering sustainable water from wastewater. Environmental S cience and T echnology , 38 (11), 201A - 208A. Li, J., Yang, F., Li, Y., Wong, F. S., & C hua, H. C. (2008). Impact of biological constituents and properties of activated sludge on membrane fouling in a novel submerged membrane bioreactor. Desalination , 225 (1), 356 - 365. Li, H., Yang, M., Zhang, Y., Liu, X., Gao, M., & Kamagata, Y. (2004). Compa rison of nitrification performance and microbial community between submerged membrane bioreactor and conventional activated sludge system. Water S cience and T echnology: A J ournal of the International Association on Water Pollution Research , 51 (6 - 7), 193 - 200 . Li, F., Wichmann, K., & Otterpohl, R. (2009). Review of the technological approaches for grey water treatment and reuses. Science of the Total Environment , 407 (11), 3439 - 3449. Liu, R., Huang, X., Liu, R., & Qian, Y. (2001). A Comparison between a Submerg ed Membrane Bioreactor and a Conventional Activated Sludge Process. Chinese Journal OF Environemntal Science , 22 (3), 20 - 24. Madaeni, S. S., Fane, A. G., & Grohmann, G. S. (1995). Virus removal from water and wastewater using membranes. Journal of Membrane S cience , 102 , 65 - 75. Marrot, B., Barrios Martinez, A., Moulin, P., & Roche, N. (2004). Industrial wastewater treatment in a membrane bioreactor: a review. Environmental P rogress , 23 (1), 59 - 68. Melin, T., Jefferson, B., Bixio, D., Thoeye, C., De Wilde, W., De Koning, J., ... & Wintgens, T. (2006). Membrane bioreactor technology for wastewater treatment and 92 reuse. Desalination , 187 (1), 271 - 282. Meng, F., Shi, B., Yang, F., & Zhang, H. (2007). Effect of hydraulic retention time on membrane fouling and biomass characteristics in submerged membrane bioreactors. Bioprocess and B iosystems E ngineering , 30 (5), 359 - 367. Meng, F., Shi, B., Yang, F., & Zhang, H. (2006a). New insights into membrane fouling in submerged membrane bioreactor based on rheology and hydrodynam ics concepts. Journal of M embrane S cience , 302 (1), 87 - 94. Meng, F., Zhang, H., Yang, F., Li, Y., Xiao, J., & Zhang, X. (2006b). Effect of filamentous bacteria on membrane fouling in submerged membrane bioreactor. Journal of Membrane Science , 272 (1), 161 - 168 . Miller, W. G. (2006). Integrated concepts in water reuse: managing global water needs. Desalination , 187 (1), 65 - 75. Munz, G., Gualtiero, M., Salvadori, L., Claudia, B., & Claudio, L. (2008). Process efficiency and microbial monitoring in MBR (membrane bi oreactor) and CASP (conventional activated sludge process) treatment of tannery wastewater. Bioresource T echnology , 99 (18), 8559 - 8564. Murase, T., Ohn, T., & Kimata, K. (1995). Filtrate flux in crossflow microfiltration of dilute suspension forming a highl y compressible fouling cake - layer. Journal of M embrane S cience , 108 (1), 121 - 128. Nagaoka, H., Ueda, S., & Miya, A. (1996). Influence of bacterial extracellular polymers on the membrane separation activated sludge process. Water Science and Technology , 34 (9 ), 165 - 172. National Water Research Institute, (2012). Direct Potable Reuse: Benefits for Public Water Supplies, Agriculture, the Environment, and Energy Conservation. Publication Number NWRI - 2012 - 01. National Academy of Sciences, (2012). Understanding Wat er Reuse: Potential for Expanding the Nation's Water Supply Through Reuse of Municipal Wastewater. ISBN 978 - 0 - 309 - 26520 - 1. http://www.nap.edu/catalog.php?record_id=13514 National Research Counci l, (2012). Water Reuse: Potential for Expanding the Nation's Water Supply Through Reuse of Municipal Wastewater . Washington, DC: The National Academies Press. Oehmen, A., Zeng, R. J., Yuan, Z., & Keller, J. (2005). Anaerobic metabolism of propionate by pol yphosphate accumulating organisms in enhanced biological phosphorus removal systems. Biotechnology and B ioengineering , 91 (1), 43 - 53. Oehmen, A., Lemos, P. C., Carvalho, G., Yuan, Z., Keller, J., Blackall, L. L., & Reis, M. A. (2007). Advances in enhanced biological phosphorus removal: from micro to macro scale. Water R esearch , 41 (11), 2271 - 2300. Ognier, S., Wisniewski, C., & Grasmick, A. (2002). Membrane fouling during constant flux filtration in membrane bioreactors. Membrane Technology , 2002 (7), 6 - 10. 93 Ognier, S., W isniewski, C., & Grasmick, A. (2004). Membrane bioreactor fouling in sub - critical filtration conditions: a local critical flux concept. Journal of Membrane Science , 229 (1), 171 - 177. Ou yang, K., & Liu, J. X. (2009). Analysis of characteristics of microbial communities in membrane bioreactor and conven tional activated sludge process . Huan jing ke xue= Huanjing kexue/[bian ji, Zhongguo ke xue yuan huan jing ke xue wei yuan hui" Huan jing ke xue" bian ji wei yuan hui.] , 30 (2), 499 - 503. OVIVO case study, (2011). Delphos, OH, Wastewater Treatment Plant. http://www.ovivowater.us/content/files/data/Delphos%20Case%20Study_d76b4cbe572a45c1 94bcb622e262 bae5.pdf Pauwels, B., Fru Ngwa, F., Deconinck, S., & Verstraete, W. (2006). Effluent quality of a conventional activated sludge and a membrane bioreactor system treating hospital wastewater. Environmental T echnology , 27 (4), 395 - 402. Pellegrin, M. L., Hatc her, J. (2008). Old Plant, New Tricks - MBR Retrofit: How to Maximize Performance While Minimizing Costs & Distruptions. Florida Water Resources Journal. Pescod, (1992). Wastewater treatment and use in agriculture. FAO irrigation and drainage paper 47. ISB N 92 - 5 - 103135 - 5. Reid, E., Liu, X., & Judd, S. J. (2006). Effect of high salinity on activated sludge characteristics and membrane permeability in an immersed membrane bioreactor. Journal of Membrane Science , 283 (1), 164 - 171. Rittmann, B. E., Bae, W., Namk ung, E., & Lu, C. J. (1987). A critical evaluation of microbial product formation in biological processes. Water Science and Technology , 19 (3 - 4), 517 - 528. Rosenberger, S., Krüger, U., Witzig, R., Manz, W., Szewzyk, U., & Kraume, M. (2002). Performance of a bioreactor with submerged membranes for aerobic treatment of municipal waste water. Water Research , 36 (2), 413 - 420. Saddoud, A., Ellouze, M., Dhouib, A., & Sayadi, S. (2006). A comparative study on the anaerobic membrane bioreactor performance during the treatment of domestic wastewaters of various origins. Environmental T echnology , 27 (9), 991 - 999. Saddoud, A., Ellouze, M., Dhouib, A., & Sayadi, S. (2007). Anaerobic membrane bioreactor treatment of domestic wastewater in Tunisia. Desalination , 207 (1), 205 - 215. Saddoud, A., Abdelkafi, S., & Sayadi, S. (2009). Effects of domestic wastewater toxicity on anaerobic membrane bioreactor (MBR) performances. Environmental T echnology , 30 (13), 1361 - 1369. Sato, T., & Ishii, Y. (1991). Effects of activated sludge proper ties on water flux of ultrafiltration membrane used for human excrement treatment. Water Science and Technology , 23 (7 - 9), 1601 - 1608. Shin, H., & Kang, S. (2003). Performance and membrane fouling in a pilot scale SBR process coupled with membrane. Water Sci ence and Technology , 47 (1), 139 - 144. 94 Silva, C. C., Jesus, E. C., Torres, A. P., Sousa, M. P., Santiago, V. M., & Oliveira, V. M. (2010). Investigation of bacterial diversity in membrane bioreactor and conventional activated sludge processes from petroleum refineries using phylogenetic and statistical approaches. Journal of M icrobiology and B iotechnology , 20 (3), 447 - 459. Sima, L. C., Schaeffer, J., Le Saux, J. C., Parnaudeau, S., Elimelech, M., & Le Guyader, F. S. (2011). Calicivirus removal in a membrane bi oreactor wastewater treatment plant. Applied and E nvironmental M icrobiology , 77 (15), 5170 - 5177. Simmons, F. J., and Xagoraraki, I. (2011). Release of infectious human enteric viruses by full - scale wastewater utilities. Water R esearch , 45: 3590 - 3598. Simmon s F. J., Kuo, D. H. - W., Xagoraraki, I. (2011). Removal of human enteric viruses by a full - scale membrane bioreactor during municipal wastewater processing. Water R esearch , 45: 2739 - 2750. Soriano, G. A., Erb, M., Garel, C., & Audic, J. M. (2003). A comparat ive pilot - scale study of the performance of conventional activated sludge and membrane bioreactors under limiting operating conditions. Water E nvironment R esearch , 225 - 231. State Water Resources Control Board, (2013). Recycled Water Policy. Resolution 2013 - 0003. Tazi - Pain, A., Schrotter, J. C., Bord, G., Payreaudeau, M., & Buisson, H. (2002). Recent improvement of the BIOSEP® process for industrial and municipal wastewater treatment. Desalination , 146 (1), 439 - 443. United States Environmental Protection Agen cy, (2007). Wastewater Management Fact Sheet. https://www.google.com/#q=Wastewater+Management+Fact+Sheet++1++Membrane+B ioreactors United States Environm ental Protection Agency, (2012). Guidelines for Water Reuse. EPA/600/R - 12/618. Visvanathan, C., Yang, B. S., Muttamara, S., & Maythanukhraw, R. (1997). Application of air backflushing technique in membrane bioreactor. Water S cience and T echnology , 36 (12), 259 - 266. Wachinski, M. T. (2013). Membrane process for water reuse. McGraw Hill Companies, Inc. ISBN 0 - 07 - 174895 - 4. Wang, L. K., Shammas, N. K., & Hung, Y. T. (2009). Handbook of Environmental Engineering Series, Vol. 9, Advanced Biological Treatment Proce sses. Human Press. ISBN: 978 - 1 - 58829 - 360 - 2, DOI: 10.1007/978 - 1 - 60327 - 170 - 7. Water Reuse Association, (2009). Potable Reuse Program Position Statement. http:// www.watereuse.org/sites/default/files/u8/PR%20position%20statement%20v3a.pd f Water Reuse Association, (2009b). WateReuse Association 2008 Property Owner and Homeowner Market Perception Study: Final Report. Group 3 Research, Pittsburg, PA. Water Reuse Asso ciation, (2010). Direct Potable Reuse Workshop. http://www.nwri - usa.org/pdfs/DirectPotableWorkshopSummaryFINAL091010.pdf 95 Wei, Y., van Houten, R. T., Borger, A. R., Eik elboom, D. H., & Fan, Y. (2003). Comparison performances of membrane bioreactor and conventional activated sludge processes on sludge reduction induced by Oligochaete. Environmental S cience and T echnology , 37 (14), 3171 - 3180. Wen, X., Ding, H., Huang, X., & Liu, R. (2004). Treatment of hospital wastewater using a submerged membrane bioreactor. Process B iochemistry , 39 (11), 1427 - 1431. Winward, G. P., Avery, L. M., Frazer - Williams, R., Pidou, M., Jeffrey, P., Stephenson, T., & Jefferson, B. (2008). A study of the microbial quality of grey water and an evaluation of treatment technologies for reuse. Ecological Engineering , 32 (2), 187 - 197. World Health Organization, (1989). Health guidelines for the use of wastewater in agriculture and aquaculture. Technical Repo rt Series 778. ISBN 92 4 120778 7. Wu, J., Li, H., & Huang, X. (2010). Indigenous somatic coliphage removal from a real municipal wastewater by a submerged membrane bioreactor. Water R esearch , 44 (6), 1853 - 1862. Xagoraraki, I., Yin, Z., & Svambayev, Z. (201 4). Fate of Viruses in Water Systems. Journal of Environmental Engineering . Zanetti, F., De Luca, G., & Sacchetti, R. (2010). Performance of a full - scale membrane bioreactor system in treating municipal wastewater for reuse purposes. Bioresource T echnology , 101 (10), 3768 - 3771. 96 CHAPTER 3 HUMAN ADENOVIRUS REMOVAL BY HOLLOW FIBER MEMBRANES: EFFECT OF MEMBRANE FOULING BY SUSPENDED AND DISSOLVED MATTER Abstract Virus removal in membrane bioreactors is of concern since membrane pore size can be larger than t he size of certain viruses. In this study, we evaluated removal of human adenovirus 40 (HAdV 40) by hollow fiber ultrafiltration (UF, microfiltration (MF1, the constant flux regime and in the presence of aeration. Individual and combined impacts of suspended (SiO 2 microspheres) and dissolved (Aldr ich humic acid) foulants on permeate flux and virus removal were determined and compared. Average removal of HAdV 40 from DI water by UF, MF1 and MF2 membranes was 2.3 log, 0.7 log and 0.7 log, respectively. The observed decrease in HAdV 40 removal due to SiO 2 - 1.2 and - 0.2 for UF and MF1 respectively) was attributed to the cake - enhanced accumulation of viruses at the 0.8 and 1.2 for UF and MF1, respectiv ely), which was attributed to pore blockage by humic acid. In experiments with MF2 membrane, neither humic acid nor SiO 2 had statistically significant effects on HAdV 40 removal. Combined fouling by humic acid and SiO 2 led to HAdV 40 removal that appeared to a superposition of individual contributions of these constituents. The results indicate that the extent of fouling is not a reliable predictor of virus removal. Instead, feed water composition and membrane pore size together govern virus removal with fo uling mechanisms playing a key mediating role: pore blockage improves virus removal while cake formation can either increase or decrease virus removal depending on cake properties. 97 Keywords: microfiltration, ultrafiltration, membrane fouling, virus remova l, adenovirus 98 3. 1. Introduction More than 150 types of enteric viruses have been found in contaminated waters (Leclerc et al. 2000; Wong et al. 2012; Havelaar et al. 1993; Melnick et al. 1978). Because of their low infectious dose and long survival in the environment viruses pose a considerable th reat to human health. Human adenovirus (HAdV) is one of enteric viruses on the U.S. EP contaminant candidate list. Various species of HAdV can cause a range of diseases ( Heim et al. 2003; Jones et al. 2007) ; fo r example, HAdV - F is the known etiological agent of gastroenteritis while HAdV - B and HAdV - E may lead to acute respiratory diseases. A double - strand DNA virus, HAdV is one of largest virions ranging from 70 to 140 nm in size (Xagoraraki et al. 2014). What m akes HAdV particularly problematic is its resistance to UV disinfection (K o et al. 2005; Nwachuku et al. 2005; Baxter et al. 2007) with UV dosages as high as 217.1 mW/cm 2 required for 99.99% deactivation of HAdV 40 (Thurston - Enriquez et al. 2003). The larg e size of HAdV and its resistance to UV light point to the promise of membrane filters as a treatment process for removing this virus from water. Although some pathogen removal occurs during wastewater treatment, even advanced technologies may not provide an absolute barrier for viruses. Indeed, recent studies report presence of human enteric viruses in the effluents of state - of - the art treatment facilities such as membrane bioreactors (MBR) plants (Kuo et al. 2010; Simmons et al. 2011) and drinking water t reatment plants (Sedmak et al. 2005; Albinana - Gimenez et al. 2009) . MBRs can achieve high and stable removal efficiency for chemical oxygen demand (Pankhania et al. 1994; Beaubien et al. 1996) , biochemical oxygen demand (Kishino et al. 1996) , nitrogen (Ki s hino et al. 1996; Gujer et al. 1999), phosphorus (Schaum et al. 2005) , and coliform bacteria (Van der Roest et al. 2002). Virus removal, however, has not been a criterion in the design and operation of MBR plants. In fact, some MBRs employ membranes with the nominal pore size larger than the size of a typical virus (20 200 nm), in which case 99 membrane fouling and cleaning may control virus removal. Multiple studies evaluated virus removal as a function of membrane and feed properties; some of this work h as employed bacterioph ages as human virus surrogates. Langlet et al. showed an increase in MS - 2 phage removal with a decrease in the membrane pore sizes (Langlet et al. 2009) . Lu et al. (2013) found a strong linear correlation between MS - 2 log removal and permeability of ultrafilters in the presence of foulants in the feed: on average, fouling increased MS - 2 removal by 1.23 logs . Working with the same type of phage, Jacangelo et al. reported that membrane fouling contributed up to 2.6 logs removal of MS - 2, which was much more significant comparing to physical sieving/adsorption (0.3 log) and cake layer formation (0.1 0.5 log) (Jacangelo et al. 1995) . Wu et al. (2010) reported that gel layer contributed to the removal of somatic coliphage removal, more so a t a higher permeate flux. High removals of T4 coliphage have been reported and partly attributed to the formation of a cake layer formed on membrane surface (Lv et al. 2006; Zh eng et al. 2005; Zheng and Liu 2007). Shirasaki et al. (2008) carried out filtra tion experiments in a coagulation MF system and concluded that irreversible fouling played a more important role than reversible fouling in enhancing virus removal . Farahbakhsh and Smith (2004) investigated coliphage removal from secondary effluent of wast ewater treatment plant by microfiltration membrane and reported that fouled membranes reje cted viruses more effectively. Composition of the feed water (pH, ionic strength, presence of divalent actions and organic matter) and pretreatment were suggested as key factors governing virus removal (van Voorthuizen et al. 2001; Huang et al. 2012; Fiksdal and Leiknes 2006; Madaeni et al. 1995; Matsushita et al. 2005; Zhu et al. 2005) . To our knowledge, there have been only six studies on adenovirus removal by membra nes with all this work performed in the context of MBR treatment. Sedmak et al. effluent although in a much smaller fraction of samples and much lower titers than in th e 100 influent (Sedmak et al. 2005). Albinana - Gimenez et al. (2009) reported sporadic qPCR - positive but PFU - negative results in the effluent from drinking water treatment plants . (In contrast, culturable HAdV in MBR effluent was measured in effluents of each o f 10 conventional WWTPs sampled by Hewitt et al. (2011)). Kuo et al. (2010) showed that HAdV species A, C, and F were removed only partially in the Traverse City MBR WWTP and showed that with the average HAdV removal of 5.0 0.6 logs over the 8 month long s tudy, the effluent contained on average ~ 10 3 HAdV particles/L . In their study of enteric virus removal in conventional WWTPs and microfiltration MBR WWTPs (equipped with Kubota membranes), Francy et al. (2012) showed that HAdV was detected by q - PCR in a s ubset of MBR effluent samples both before and after UV disinfection . In a survey of virus removal in nine MBR WWTPs employing different kinds of membranes (tubular, hollow fiber and flat sheet; MF and UF) , Hirani et al. (2013) reported that adenoviruses we re detected in effluents of all MBR facilities sampled; this result was consistent with the findings by Kuo et al. (2010) and was particularly striking because enteroviruses, rotaviruses and hepatitis A viru ses were absent in all samples. The authors tenta tively attributed this finding to the fact that HAdV and risk assessment on presence of adenovirus in In a follow - up study (Hirani et al. 2014) with four different membrane systems, these authors showed that adenoviruses were always detected in MBR filtrate samples by PCR regardless of whether the membrane was breached (effluent turbidity > 0.5 NTU) or cleaned (0.2% NaClO). The objective of the present work was to elucidate mechanisms of HAdV removal by membranes in the pre sence of foulants in the feed. To facilitate mechanistic insights, we employed two well characterized model foulants (humic acid and silica particles), three commercial ly available hollow fiber membranes (with pore sizes typical for membranes used in MBRs), and filtration conditions that matched, to the extent possible, the protocol used at 101 full - scale MBR facilities (i.e. constant flux regime, aeration). 3. 2. Materials and Methods 3.2.1. Cell culture experiment and virus incubation A549 cell line has been suggested as an efficient cell line for HAdV (Witt and Bousquet, 1988; Lee et al., 2004), and it was selected in this study. A549 cells (ATCC, cell passage were incubat ed at 37°C with growth medium (minimum essential medium with 10% fetal bovine serum, L - was discarded from the flask, and HAdV 40 was added and incubated at 37°C with growth me dia (2% fetal bovine serum) until cytopathic effect became apparent. In order to isolate viruses from cell debris, virus suspension was centrifuged at 400 g for 4 min, and then - driven filter (Millipore). Filtered virus stoc k suspension had HAdV concentration of approximately 10 10 copies/mL and was stored at - 80°C before use. 3.2.2. Membrane preparation Three types of ho llow fiber membranes were used in this study. The characteristics of the membranes are shown in Table 3 - 1 . The hollow fibers were cut into 80 cm long segments and assembled by looping and potting the using an adhesive (Loctite). Each membrane bundle (12 loops of 0. cm 2 . After the adhesive dried, membranes were soaked in DI water for at least 24 h before use. 102 3.2.3. Foulant preparation and particle size Silica m icrospheres and humic acid (HA) were selected as model foulants. According to the manufacturer, the average particle size of spherical SiO 2 (99.998% purity, Nanostructured & Amorphous Materials) was in the 1 to 3.5 µm range. To prepare a feed suspension wi th silica, SiO 2 particles were added to 0.5 L of DI water, mixed for 1 h and then added to the feed tank. To prepare a feed suspension with HA, 12 g of HA (Aldrich) were added into 4 L of DI water in an amber jar and the pH was adjusted to 8. The solution was mixed using a Filtered HA solution was stored at 4°C until use. Total organic carbon (TOC) content of the feed water was measured using TOC analyzer (OI Analyti cal). Particle size distribution in the stock was measured using Mastersizer 2000 (Malvern). 3. 2. 4. Membrane filtration experiment The schematic of the experimental unit is shown in Figure 3 - 1 . The total volume of the reactor was 25 L. Diffusers were plac ed at the bottom of the feed tank to supply air and mix the feed water. Peristaltic digital pump (model 07523 - 80, MasterFlex L/S) was used to apply transmembrane pressure. Permeate flow rate and transmembrane pressure were measured using a digital flow met er (model 106 - 4 - C - T4 - C10, McMillan) and digital pressure sensor ( Cole - Parmer, 68075 - 00 ), respectively. A LabView code was developed to record readings from the flowrate and pressure sensors and to control the flow rate of the pump. Three experiments with f eeds of different compositions were carried out with each type of hollow fiber membranes. Each experiment included 4 stages: Stage 1 (duration = 1 h). The feed tank, with air diffusers on the bottom, was filled with 18 L of DI water and 10 mL of HAdV 40 stock suspension was added to the DI water in the tank. Averaged over all experiments, the initial feed concentration of HAd V 40 in 103 the tank was 7.03 ± 0.32. The pH of the feed was adjusted to 7. Stage 2 (duration = 6 h). The transmembrane pressure was applied and filtration was carried out in a constant flux regime ( = 50 mL/min; = 2 .78·10 - 5 m/s). Samples of feed and permeate were withdrawn periodically for qPCR analysis and calculation of HAdV rejection. Stage 3 (duration = 8 h). Foulants were added to the feed tank. pH was adjusted to 7 again. Stage 4 (duration = 6 h for UF membranes and 12 h for MF membranes). Transmembrane pressure was applied and the fouling test was carried out in constant flux regime ( Q = 50 mL/min; j = 2.78·10 - 5 m/s). As in Stage 2, samples of feed and permeate were withdrawn p eriodically for qPCR analysis and calculation of HAdV rejection. At each stage the feed water was mixed by continuous aeration. Feed and permeate samples wer e taken when the flow rate reached the target value of 50 mL/min during Stage 2, and every 2 h afte rward. All feed and permeate samples withdrawn from the feed tank during this stage were stored at - 80 °C until DNA extraction. The sampling protocol for each experiment is detailed in Table 3 - 2. The high foul ant concentrations were used to accelerate memb rane fouling and shorten the time of data gathering. 3. 2. 5. DNA extraction and quantitative polymerase chain reaction (qPCR) Virus DNA in each sample was extracted using MagNa Pure Compact System automatic machine and Nucleic Acid Isolation Kits (Roche Ap plied Sciences). Carrier RNA (Qiagen) was used to enhance DNA recovery. The DNA extracts were placed into storage ( - 80 °C) immediately after extraction. Following DNA extraction, virus concentration was quantified using qPCR (Roche Light Cycler). Sequence of primers and TaqMan probe were adopted from Xagoraraki et al. (2007). Values of crossing point, C p , were automatically generated by 104 the LightCycler software. HAdV concentrations in feed and permeate sample were determined based on the C p values and the standard curve that was developed beforehand. 3.2.6. Inhibition of qPCR by humic acid reported that polymerase chain reaction may be inhibited by HA. In order to evaluate the effect of qPCR inhibitors, we ada pted the method of serial dilutions that was used by Ijzerman et al. (1997) and Gibson et al. (2012). A set of HA solutions with different concentrations of HA (0, 10, 20, 30, and 40 mg/L) were seeded with ~10 7 copies/mL of HAdV 40. Then DNA extraction and qPCR analysis were carried out to assess HA - induced inhibition of qPCR. 3.2.7. Scanning electron microscopy (SEM) imaging of membranes SEM images of membrane skin surfaces and cross - sections were recorded using JSM - 7500F microscope . When imaging skin sur faces of UF, MF1, and MF2 membranes, the magnifications were ×100,000, ×10,000, and ×2,200 respectively. Images of membrane cross - sections were taken with the magnification of ×170. SEM samples were prepared by immersing membrane coupons in liquid N 2 , brea king them into smaller fragments, and mounting them on SEM aluminum stubs. MF1 and MF2 membranes were coated with ~ 14 nm Au layer in the Emscope sputter coater, while UF membranes were coated with ~ 4 nm Pt layer in the Electron Microscopy Sciences Q150T turbo - pumped coater. 3.2.8. Membrane challenge tests To quantitative assess the retention ability of the membranes and supplement nominal pore size data provided by the manufacturers (Table 3 - 1), membrane challenge tests were performed using suspensions o f monodisperse spheri cal probe particles. Fluorescent 105 polystyrene beads with the nominal diameter of 50, 100, 300, and 500 nm (PSF series) were purchased from Magsphere, Inc. The challenge tests were performed using the same filtration rig (except that a 1 L Nalgene bottle was used as a feed vessel) as in virus filtration studies ( S ection 2 .2. 4) and were run for 15 min at the constant permeate flow rate of 50 mL/min. For each probe/membrane combination, three permeate samples were collected 11, 13, and 15 m in into the challenge test and the log removal (or rejection) value was calculated as an average for these three samples. Particle concentrations in the feed and permeate were determined spectrophotometrically (Multi - Spec 1501, Shimadzu). The absorbance wa s measured at = 197, 202, 236, and 274 nm with 50, 100, 300, and 500 nm probes, respectively. 3. 3. Results and Discussion 3.3.1. Characterization of membranes and model foulants Particle size distributions for SiO 2 suspension and solution o f humic acid are shown in Figure 3 - 2. There was approximately an order of magnitude difference in size between the silica microspheres and humic acid aggregates. Values of d 0.1 , d 0.5 , and d 0.9 for the suspension SiO 2 par ticles were 1.81, 3.45, and 6.81 µm, respectively while for humic acid these values were 0.09, 0.15, and 0.33 µm. Results of membrane challenge tests (Table 3 - 3) were consistent with the nominal pore sizes reported by the manufacturers (Table 3 - 1) and resu lts of SEM imaging (Figure 3 - 3). As expected, larger probes were rejected more by all three membranes. The 100 nm probe, which was the closest to the size of HAdV - 40, was rejected by UF, MF1 and MF2 me mbranes with rejections of 97.5%, 84.2% and 82%, which corresponded to LRV values of 1.61, 0.80, and 0. 75 (Table 3 - 3). 10 6 3.3.2. Inhibition of qPCR by humic acid HA - free and 10, 20, 30, and 40 mg (TOC)/l solutions of HA seeded with 10 7 copies/mL (i.e. 7 logs) of HAdV 40 were analyzed for virus concentration. In samples with 0, 10, 20, and 30 mg(TOC)/l, the concentration of HAdV was measured to be 7.02, 6.95, 6.93 and 6.82 logs, respectively. Only, in the 40 mg (TOC)/l solution the virus concentration could not be measured apparently because the fluorescence signa l during Light Cycler measurements was inhibited by organic compounds in the sample. Given the negligible inhibition at sufficiently low HA concentrations, all feed samples were diluted ten - fold to adjust HA concentration to 4 mg/L. Additionally, permeate samples from experiments with 0.22 µm and 0.45 µm membranes treating the mixture of SiO 2 and HA were also diluted. As a control measure, all original and diluted samples were analyzed for comparison. Paired t - test showed significant (p < 0.05) difference b etween the original and adjusted concentrations in feed samples. In contrast, no significant difference was found for permeate samples. The dilution factor was taken into account during the virus removal calculation afterward. 3.3.3 . Membrane fouling and transmembrane pressure buildup Figure 3 - 3 illustrates changes i n the transmembrane pressure with filtration time. During Stage 2 (filtration of HAdV 40 in DI water), the headloss increased slowly: was only 0.05, 0.04, and 0.07 psi/h (345, 275 , and 483 Pa/h) on average in filtration tests with UF ( = 0.04 µm), MF1 ( = 0.22 µm), and MF2 ( = 0.45 µm) membranes, respectively. This was due to the relatively low concentration of HAdV 40, the only foulant in the feed. Thus neither complete pore blocking (more likely to occur with the UF membrane), nor standard blocking (more likely to occur with the MF2 membrane) by HAdV 40 added much additional re sistance to the permeate flow. In these constant flux experiments, t he 107 permeate flow rate was maintained at 49.97 ± 1.60, 49.99 ± 1.44, and 49.17 ± 4.51 mL/min, respectively. During Stage 4 of experiments with all three types of membranes, fouling by the mixture of HA and SiO 2 particles was significantly higher than the sum of contributions due to each of these two feed components fouling the membrane separately. For example, for the UF membrane ( = 0.04 µm), the pressure increase rate during initial stages of fouling was ~ 1.3, 1.4, and 24.5 psi/h (9.0, 9.7, and 175.2 kPa/h) on average in tests with SiO 2 , HA, and SiO 2 /HA m ixture, respectively. We hypothesize that the synergy stemmed from two separate but related effects. On the one hand, accumulation of SiO 2 particles on the membrane surface likely hindered b ack - diffusion of HA away from the membrane and resulted in more blockage of membrane pores by HA. On the other hand, such accumulation of HA near the membrane in the presence of a SiO 2 cake, could lead to the formation of a composite SiO 2 /HA layer with a h igher specific hydraulic resistance than that of the cake composed of SiO 2 particles only. Consistent with the above hypothesis is the observation that the mutual enhancement of fouling by SiO 2 and HA was particularly evident in experiments with the UF mem brane; indeed, HA should be more effective in blocking smaller pores of this membrane. To confirm that pore blockage and cake formation were indeed operative fouling mechanisms, we performed a separate set of constant pressure dead - end filtration experimen ts and applied blocking laws (Hermia 1982) to the permeate flux data generated in these tests. The tests were performed in the absence of aeration to satisfy assumptions behind f the same size was not met though: UF, MF1, and MF2 are phase inversion membranes with complex only approximations. Figure 3 - 5 presents an example of filtration data (Stage 4; see S ection 3.2. 4) in the vs format where three segments corresponding to pore blockage (0 108 < < 2) and cake filtration (= 0) and the transition between these two regimes ( < 0) could be discer ned. However, it was also clear that applying one of the three blocking laws does not provide a complete description of the fouling process as segments of the vs dependence had negative slope not accounted for by the model. The negative slope is explained by the combined pore blockage - cake filtration model (Ho and Zydney, 2000) as resulting from the simultaneous pore blockage and formation of the cake over blocked areas of the membrane. The observed negative slope is consistent with the findings of Yuan et al. (2002) who suggested that membrane fouling by humic acid is a combined effect of pore blockage and cake layer formation. While the slope for the MF2 membranes was somewhat variable and deviated from zero, the vs than that recorded for the UF membrane. Figure 3 - 5 confirms that the main mechanism of fouling by SiO 2 was cake filtration and that pore blockage was one of fouling mechanisms during UF filtration of HA - containing feed waters. 3.3.4 . Removal of human adenovirus 40 by clean and fouled membranes As shown in Figure 3 - 6, the removal of virus suspended in DI water (Stage 1) by UF ( - test showed that virus removal by the UF membrane was significantly (p < 0.05) higher than by each of the MF membranes, while there was n o significant difference in virus removal between MF1 and MF2. The standard deviations reflect the variability in virus rejection with time. 109 with size exclusion as a re moval mechanism. That HAdV 40 is not completely removed by the UF membrane can be attributed to the finite width of the pore sizes distribution of the membrane. In a survey that included 27 membranes and two phages (Qâ and T4), Urase et al. (1996) reported incomplete removal of viruses for all membranes (including track etched and other narrow pore distribution ultra - and microfilters) and attributed this result to the presence of abnormally large pores. Figure 3 - 7 describes virus removal by UF, MF1, and MF 2 in the presence of foulants in the feed. Values of log removal of HAdV 40 from DI water (Stage 2) are shown next to removal values from Stage 4 of the same experiment. We also note that removal data in Figure 3 - 6 are averages of the values of HAdV remova l from DI water as reported in Figure 3 - 7; for example, the LRV value of 2.27 given in Figure 3 - 6 for the UF membrane is the average of the three values (LRV = 2.23, 1.96, and 2.62) reported in Figure 3 - 7a. Virus removal by UF membrane ( = 0.0 4 µm) was e nhanced in the presence of HA as the only foulant but was reduced when SiO 2 particles were the sole foulant (Figure 3 - 7a). These opposite effects can be tentatively attributed to two different mechanisms. The first mechanism is the partial or c omplete blockage of membrane pores by HA. Partial blockage permeation of water but not virus passage. (Incidentally, the fouling model developed by Ho and Zydney (2000) a llows some fluid flow through blocked pores.) Either scenario be it partial or complete blockage leads to improved virus removal. Competition for the adsorption sites on the membrane surface could be a contributing factor: previous studies have demonst rated that HA inhibits virus adsorption onto membranes and decreases their ability to retain viruses during filtration (Zheng et al., 2005; Zheng and Liu, 2007). The second mechanism is the possible increase of the transmembrane differential in virus conce ntration due to cake formation. The latter mechanism is consistent with the hypothesis 110 proposed in S ection 3.3. 3 where permeate flux decline was interpreted in terms of the cake - enhanced concentration polarization and accumulation of HA in the pores of bot h the membrane and the SiO 2 layer. The cake composed of larger SiO 2 particles is too porous to effectively reject HAdV 40 yet may be sufficiently dense to hinder HAdV 40 back - diffusion leading to its accumulation near the cake - membrane interface. The highe r transmembrane differential of virus concentration leads to enhanced virus transport across the membrane and lower virus removal. When both foulants are present in the feed, the two opposing effects appear to cancel each other (Figure 3 - 7a). In experiment s with MF1 ( = 0.22 µm) the effects of the two types of foulants acting alone and in combination were similar to but less pronounced than those observed in UF tests (Figure 3 - 7b). In experiments with MF2 membrane ( = 0.45 µm), howev er, neither HA alone nor SiO 2 alone had statistically significant effects on HAdV 40 removal (Figure 3 - 7c). HA molecules were not large enough to block membrane pores effectively; indeed, HA was pre - filtered through a 0.45 ìm membrane prior to virus remova l tests (see S ection 3.2. 3). Formation of the SiO 2 - only cake lead to less significant cake - enhanced accumulation of the virus and had only a slight effect on virus removal by MF2 membranes. This was because the effective pore size of the cake was closer to that of the MF2 skin (than to that of UF and MF1 skins); thus, fouling of MF2 by SiO 2 effectively increased the thickness of the membrane, added a small additional resistance to the permeate flow (Figure 3 - 4c) but did not significantly increase virus conc entration at the membrane surface. Combined fouling of MF2 by HA and SiO 2 lead to a statistically significant decrease in HAdV 40 removal (Figure 3 - 7c). Apparently the composite HA/SiO 2 cake was dense enough to capture and concentrate viruses for higher tr ansmembrane concentration differential and enhance virus transport across the membrane; at the same time, in contrast to UF and MF1, MF2 membrane pores were too large to be blocked by HA and reject viruses. Figure 3 - 8 111 schematically illustrates how the two hypothesized effects of fouling on virus removal manifest themselves in UF (Figure 3 - 8; A, B, C) and MF (Figure 3 - 8; D, E, F): Figure 3 - 8 A, C: increased virus removal due to pore blockage by dissolved species (also see Figure 3 - 7a (HA and HA+SiO 2 data)); Figure 3 - 8 B, C: decreased removal due to cake - enhanced accumulation of virus near the membrane (also see Fig. 7a (SiO 2 and HA+SiO 2 data)); Figure 3 - 8 C, F: additional removal by the composite HA/SiO 2 cake (also see Fig ure 7c (HA+SiO 2 data)). It is very clear fr om Figure 3 - 7 and Figure 3 - 4 that the extent of fouling or rate of fouling increase are not reliable predictors of virus removal, which can either increase or decrease as a result of membrane fouling. Instead, feed water composition and membrane po re size together govern virus removal with fouling mechanisms playing a k ey mediating role: pore blockage improves virus removal while cake formation can either increase or decrease virus removal depending on the permeability of the cake. 3. 4. Conclusions This study demonstrates that membrane fouling may have a profound impact on HAdV 40 removal by membranes. In the absence of fouling, average values of HAdV 40 removal by hollow fiber ultrafiltration (UF, = 0.22 log and 0.7 log, respectively. Fouling by humic acid (model dissolved species), SiO 2 microspheres (model suspended species) and a mix of separate effects are identified: 1) increased removal due to pore blockage by dissolved species; 2) decreased removal due to cake - enhanced accumulation of viruses near membrane 112 surface; and 3) increased removal by the composite cake acting as a secondary membrane. The results indicate that the extent of fouling is not a reliable predictor of virus removal. Instead, feed water composition and membrane pore size together govern virus removal w ith fouling mechanisms playing a key mediating role: pore blockage improves virus removal while cake formation can either increase or decrease virus removal depending on the relative permeability of the cake. 113 APPENDIX 114 Table 3 - 1. Charact eristics of hollow fiber membranes Membrane type Notation UF MF1 MF2 Manufacturer General Electric Shenzhen Youber Technology. Material Polyvinylidene fluoride Nominal pore size, µm 0.04 0.22 0.45 Outer diameter, mm 2.0 1.3 115 Table 3 - 2. Sampling pr otocols in fouling experiments with different membranes and feed waters of different compositions Test type Foulant concentration Experiment duration, h (Time between samples, h) in tests with different membranes = 0.04 µm = 0 .22 µm = 0.45 µm SiO 2 800 mg (SiO 2 )/L 6 (2) 12 (4) 12 (4) HA 40 mg (HA)/L 6 (2) 12 (4) 12 (4) SiO 2 , HA 800 mg (SiO 2 )/L 40 mg (HA)/L 0.5 (0.08) 3 (1) 2 ( 0.5) 116 Table 3 - 3. Log removal of probe particles in challenge tests with the UF, MF1, and MF2 membranes Diameter of the probe particle, nm Membrane type (nominal pore size) = 0.04 µm = 0.22 µm = 0.45 µm 50 1.16 ± 0.01 n/a n/a 100 1.61 ± 0.01 0.80 ± 0.02 0.75 ± 0.05 300 1.86 ± 0.10 0.89 ± 0.02 0.8 6 ± 0.04 500 n/a 1.43 ± 0.23 0.95 ± 0.01 117 Figure 3 - 1. Schemati c of the experimental apparatus Notation: 1. Air compressor; 2. Air flowmeter; 3. Air diffusers; 4. Hollow fiber membrane loops; 5. Pressure gauge; 6. Peristaltic pump; 7. Flowmeter 118 Figure 3 - 2. Particle size distribution of model foulants 119 Figure 3 - 3. SEM micrographs of cross - sections (A - C) and the planar view of the separation layer (D F) of the three membranes UF with = 0.04 µm (A, D); MF1 with = 0.22 µm (B, E); and MF2 with = 0.45 µm (C, F) 120 a) b) c) Figure 3 - 4. Transmembrane pressure as a function of time during filtration of HAdV 40 suspension ( - - , - - , - - ) and HAdV - seeded feeds containing SiO 2 micros pheres ( - - ), humic acid ( - - ) , and SiO2/HA mixture ( - - ) by three membranes of different nominal pore sizes: a) 0.04 µm, b) 0.22 µm, and c) 0.45 µ m * * Circled numbers mark experimental stages (see Section 3.2. 4) 121 Figure 3 - 5. Bl ocking laws applied to fi ltration of SiO 2 microspheres and humic acid by UF and MF2 membrane s 122 Figure 3 - 6. Remo val of HAdV 40 from DI water by three membranes of different nominal pore sizes * * UF ( = 0.04 µm), MF1 ( = 0.22 µm), and MF2 ( = 0.45 µm). The values represent averages over the duration of Stage 2 of the experiment (see S ection 3.2. 4) and over all experiments with a membrane of a given pore size. 123 (a) (b) (c) Figure 3 - 7. Comparison of HAdV 40 removal from D I water, suspension of SiO 2 microspheres, solution of humic acid, and SiO 2 /HA mixture by three membranes of different nominal pore sizes: a) 0.04 µm, b) 0.22 µm, and c) 0.45 µm * * The values represent averages over the duration of Stage 4 of the expe riment (see S ection 3.2. 4) . 124 Figure 3 - 8. Schematic illustration of effects of fouling on HAdV 40 removal by ultrafiltration (A, B, C) and microfiltration (D, E, F) membranes under conditions of fouling by dissolved species (A, D), suspended par ticles (B, E) and by both of these foulants (C, F) * * HAdV 40, dissolved species and suspended species are depicted as blue dots with spikes, orange random shapes, and gray spheres, respectively . A verage values of log removal of HAdV 40 by clean UF and MF 2 membranes are 2.27 and 0.73, respectively. 125 REFERENCES 126 R EFERENCES Albinana - Gimenez, N., Miagostovich, M. P., Calgua, B., Huguet, J. M., Matia, L., & Girones, R. (2009). Analysis of adenoviruses and polyomaviruses quantified by qPCR as ind icators of water quality in source and drinking - water treatment plants. W ater R esearch , 43 (7), 2011 - 2019. Baxter, C. S., Hofmann, R., Templeton, M. R., Brown, M., & Andrews, R. C. (2007). Inactivation of adenovirus types 2, 5, and 41 in drinking water by U V light, free chlorine, and monochloramine. Journal of Environmental Engineering , 133 (1), 95 - 103. Beaubien, A., Baty, M., Jeannot, F., Francoeur, E., & Manem, J. (1996). Design and operation of anaerobic membrane bioreactors: development of a filtration te sting strategy. Journal of Membrane Science , 109 (2), 173 - 184. Francy, D. S., Stelzer, E. A., Bushon, R. N., Brady, A. M., Williston, A. G., Riddell, K. R., Borchardt, M. A., Spencer, S. K., & Gellner, T. M. (2012). Comparative effectiveness of membrane bio reactors, conventional secondary treatment, and chlorine and UV disinfection to remove microorganisms from municipal wastewaters. Water R esearch , 46 (13), 4164 - 4178. Farahbakhsh, K., & Smith, D. W. (2004). Removal of coliphages in secondary effluent by micr ofiltration - mechanisms of removal and impact of operating parameters. Water R esearch , 38 (3), 585 - 592. Fiksdal, L., & Leiknes, T. (2006). The effect of coagulation with MF/UF membrane filtration for the removal of virus in drinking water. Journal of Membr ane Science , 279 (1), 364 - 371. Gibson, K. E., Schwab, K. J., Spencer, S. K., & Borchardt, M. A. (2012). Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large - volume environmental water sampl es. Water R esearch , 46 (13), 4281 - 4291. Gujer, W., Henze, M., Mino, T., & van Loosdrecht, M. (1999). Activated sludge model no. 3. Water Science and Technology , 39 (1), 183 - 193. Havelaar, A. H., Van Olphen, M., & Drost, Y. C. (1993). F - specific RNA bacterioph ages are adequate model organisms for enteric viruses in fresh water. Applied and E nvironmental M crobiology , 59 (9), 2956 - 2962. Heim, A., Ebnet, C., Harste, G., & Pring Åkerblom, P. (2003). Rapid and quantitative detection of human adenovirus DNA by real tim e PCR. Journal of M edical V irology , 70 (2), 228 - 239. Hermia, J. (1982). Constant pressure blocking filtration law application to powder - law non - Newtonian fluid. Transaction of the Institution of Chemical Eng ineers , 60 , 183 - 187. 127 Hewitt, J., Leonard, M., Gree ning, G. E., & Lewis, G. D. (2011). Influence of wastewater treatment process and the population size on human virus profiles in wastewater. Water R esearch , 45 (18), 6267 - 6276. Hirani, Z. M., Bukhari, Z., Oppenheimer, J., Jjemba, P., LeChevallier, M. W., & Jacangelo, J. G. (2013). Characterization of effluent water qualities from satellite membrane bioreactor facilities. Water R esearch , 47 (14), 5065 - 5075. Hirani, Z. M., Bukhari, Z., Oppenheimer, J., Jjemba, P., LeChevallier, M. W., & Jacangelo, J. G. (2014). Impact of MBR cleaning and breaching on passage of selected microorganisms and subsequent inactivation by free chlorine. Water R esearch , 57 , 313 - 324. Ho, C. C., & Zydney, A. L. (2000). A combined pore blockage and cake filtration model for protein fouling during microfiltration. Journal of Colloid and Interface Science , 232 (2), 389 - 399. Huang, H., Young, T. A., Schwab, K. J., & Jacangelo, J. G. (2012). Mechanisms of virus removal from secondary wastewater effluent by low pressure membrane filtration. Journ al of Membrane Science , 409 , 1 - 8. Ijzerman, M. M., Dahling, D. R., & Fout, G. S. (1997). A method to remove environmental inhibitors prior to the detection of waterborne enteric viruses by reverse transcription - polymerase chain reaction. Journal of V irolog ical M ethods , 63 (1), 145 - 153. Jacangelo, J. G., Adham, S. S., & Laîné, J. M. (1995). Mechanism of Cryptosporidium, Giardia, and MS2 virus removal by MF and UF. Journal - American Water Works Association , 87 (9), 107 - 121. Jones, M. S., Harrach, B., Ganac, R. D ., Gozum, M. M., dela Cruz, W. P., Riedel, B., Pan, C., Delwart, E. L. & Schnurr, D. P. (2007). New adenovirus species found in a patient presenting with gastroenteritis. Journal of V irology , 81 (11), 5978 - 5984. Kishino, H., Ishida, H., Iwabu, H., & Nakano, I. (1996). Domestic wastewater reuse using a submerged membrane bioreactor. Desalination , 106 (1), 115 - 119. Ko, G., Cromeans, T. L., & Sobsey, M. D. (2005). UV inactivation of adenovirus type 41 measured by cell culture mRNA RT - PCR. Water R esearch , 39 (15), 3643 - 3649. Kuo, D. H. W., Simmons, F. J., Blair, S., Hart, E., Rose, J. B., & Xagoraraki, I. (2010). Assessment of human adenovirus removal in a full - scale membrane bioreactor treating municipal wastewater. Water R esearch , 44 (5), 1520 - 1530. Langlet, J., O gorzaly, L., Schrotter, J. C., Machina l, C., Gaboriaud, F., Duval, J. F., & membrane filtration in water treatment: applicability of real - time RT - PCR method. Journal of M embrane S cience , 326 (1), 111 - 116. 128 Leclerc, H., Edberg, S ., Pierzo, V., & Delattre, J. M. (2000). Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. Journal of A pplied M icrobiology , 88 (1), 5 - 21. Lee, C., Lee, S. H., Han, E., & Kim, S. J. (2004). Use of cell culture - PCR assay based on combination of A549 and BGMK cell lines and molecular identification as a tool to monitor infectious adenoviruses and enteroviruses in river water. Applied and E nvironmental M icrobiology , 70 (11), 6695 - 6705. Lu, R., Mosiman, D., & Nguyen, T. H. (20 13). Mechanisms of MS2 bacteriophage removal by fouled ultrafiltration membrane subjected to different cleaning methods. Environmental S cience and T echnology , 47 (23), 13422 - 13429. Lv, W., Zheng, X., Yang, M., Zhang, Y., Liu, Y., & Liu, J. (2006). Virus rem oval performance and mechanism of a submerged membrane bioreactor. Process Biochemistry , 41 (2), 299 - 304. Madaeni, S. S., Fane, A. G., & Grohmann, G. S. (1995). Virus removal from water and wastewater using membranes. Journal of Membrane Science , 102 , 65 - 75 . Matsushita, T., Matsui, Y., Shirasaki, N., & Kato, Y. (2005). Effect of membrane pore size, coagulation time, and coagulant dose on virus removal by a coagulation - ceramic microfiltration hybrid system. Desalination , 178 (1), 21 - 26. Melnick, J. L., Gerba, C. P., & Wallis, C. (1978). Viruses in water. Bulletin of the World Health Organization , 56 (4), 499. Nwachuku, N., Gerba, C. P., Oswald, A., & Mashadi, F. D. (2005). Comparative inactivation of adenovirus serotypes by UV light disinfection. Applied and E nv ironmental M icrobiology , 71 (9), 5633 - 5636. Pankhania, M., Stephenson, T., & Semmens, M. J. (1994). Hollow fibre bioreactor for wastewater treatment using bubbleless membrane aeration. Water R esearch , 28 (10), 2233 - 2236. Schaum, C., Cornel, P., & Jardin, N. ( 2005, August). Possibilities for a phosphorus recovery from sewage sludge ash. In Proceedings of the IWA Specialist Conference on Management of Residues Emanating from Wastewater Treatment (pp. 9 - 12). Sedmak, G., Bina, D., MacDonald, J., & Couillard, L. (2 005). Nine - year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a wastewater treatment plant in Milwaukee, Wisconsin (August 1994 through July 2003). Applied and Environ mental Microbiology , 71 (2), 1042 - 1050. Shirasaki, N., Matsushita, T., Matsui, Y., & Ohno, K. (2008). Effects of reversible and irreversible membrane fouling on virus removal by a coagulation - microfiltration system. Journal of Water Supply: Research and Tec hnology, Aqua , 57 (7), 501. 129 Simmons, F. J., Kuo, D. H. W., & Xagoraraki, I. (2011). Removal of human enteric viruses by a full - scale membrane bioreactor during municipal wastewater processing. Water R esearch , 45 (9), 2739 - 2750. , D., Gojanoviæ, M. D. Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid. Humic Acid , 2 , 18 - 21. Thurston - Enriquez, J. A., Haas, C. N., Jacangelo, J., Riley, K., & Gerba, C. P. (2003). Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Applied and Environmental Microbiology , 69 (1), 577 - 582. Urase, T., Yamamoto, K., & Ohgaki, S. (1996). Effect of pore structure of membranes and module configuration on virus retention. Journal of M embrane S cience , 115 (1), 21 - 29. Van der Roest, H. F., Lawrence, D. P., & Van Bentem, A. G. N. (2005). Membrane bioreactors for municipal wastewater treatment. IWA Publishing. Van Voorthuizen, E. M., Ashbolt, N. J., & Schäfer, A. I. (2001). Role of hydrophobic and electrostatic interactions for initial enteric virus retention by MF membranes. Journal of Membrane Science , 194 (1), 69 - 79. Witt, D. J., & Bousquet, E. B. (1988). Comparison of enteric adenovirus infection in various human c ell lines. Journal of V irological M ethods , 20 (4), 295 - 308. Wong, K., Fong, T. T., Bibby, K., & Molina, M. (2012). Application of enteric viruses for fecal pollution source tracking in environmental waters. Environment I nternational , 45 , 151 - 164. Wu, J., Li , H., & Huang, X. (2010). Indigenous somatic coliphage removal from a real municipal wastewater by a submerged membrane bioreactor. Water R esearch , 44 (6), 1853 - 1862. Xagoraraki, I., Kuo, D. H. W., Wong, K., Wong, M., & Rose, J. B. (2007). Occurrence of hum an adenoviruses at two recreational beaches of the great lakes. Applied and Environmental Microbiology , 73 (24), 7874 - 7881. Xagoraraki, I., Yin, Z., & Svambayev, Z. (2014). Fate of viruses in water systems. Journal of Environmental Engineering , 140 (7). Yuan , W., Kocic, A., & Zydney, A. L. (2002). Analysis of humic acid fouling during microfiltration using a pore blockage cake filtration model. Journal of Membrane Science , 198 (1), 51 - 62. Zheng, X., Wenzhou, L., Min, Y., & Junxin, L. (2005). Evaluation of viru s removal in MBR using coliphages T4. Chinese Science Bulletin , 50 (9), 862 - 867. Zheng, X., & Liu, J. (2007). Virus rejection with two model human enteric viruses in membrane bioreactor system. Science in China Series B: Chemistry , 50 (3), 397 - 404. 130 Zhu, B., Clifford, D. A., & Chellam, S. (2005). Virus removal by iron coagulation microfiltration. Water R esearch , 39 (20), 5153 - 5161. 131 CHAPTER 4 EFFECT OF PRESSURE RELAXATION AND MEMBRANE BACKWASH ON VIRUS REMOVAL IN A MEMBRANE BIOREACTOR Abstract Pressure rel axation and permeate backwash are two commonly used physical methods for membrane fouling mitigation in membrane bioreactor (MBR) systems. In order to assess the impact of these methods on virus removal by MBRs, experiments were conducted in a bench - scale submerged MBR treating synthetic wastewater. The membranes employed were hollow fibers with the nominal pore size of 0.45 µm. The experimental variables included durations of the filtration ( ), pressure relaxation ( ) and backwash ( ) steps. Both pressure relaxation and permeate backwash led to significant reductions in virus removal. For the same value of , longer filtration/relaxation cycles (i.e. larger ) led to higher transmembrane pressure ( ) but did not have a significant impact on virus removal. A shorter backwash ( = 10 min) at a higher flow rate ( = 40 mL/min) resulted in more substantial decreases in and virus removal than a longer bac kwash ( = 20 min) at a lower flow rate ( = 20 mL/min) even though the backwash volume ( ) was the same. Virus removal returned to pre - cleaning levels within 16 h after backwash was applied. Moderate to strong correlati ons ( = 0.63 to 0.94) were found between and virus removal. Keywords: membrane bioreactor, membrane fouling, human adenovirus, pressure relaxation, backwash 132 4. 1. Introduction Membrane bioreactors, a combination of activated slu dge process and membrane filtration, have developed into a staple technology for municipal and industrial wastewater treatment and a particularly attractive treatment choice for water reuse (Judd 2010) . Compared to conventional activated sludge wastewater treatment systems, MBRs are more compact and, generally, afford more stable performance (Choi et al. 2002) . With proper design and optimized operational conditions MBRs can remove a wide range of pollutants (Vaid et al. 1991 ; Pankhania et al. 1994 ; B eaubien et al. 1996 ; Kishino et al. 1996 ; Gujer et al. 1999; Van der Roest et al. 2005) . Membrane fouling in MBRs remains a major technical challenge (Bouhabila et al. 2001 ; Judd 2008 ; Cornel and Krause 2008) . Dur ing MBR operation, biosolids as well as colloidal and macromolecular species may deposit and accumulate on membrane surfaces resulting in a decline in permeate flux. A number of membrane fouling mitigation methods have been developed including pressure rel axation, air sparging and membrane cleaning by hydraulic or chemical means. Hydraulically reversible fouling is defined as fouling that can be removed by a hydraulic wash, while hydraulically irreversible fouling refers may only be removed by chemical cle aning (Chang et al. 2002) and is typically due to intrapore fouling. Air sparging mainly targets external fouling, such as a loosely attached cake layer on membrane surface while backwash can also remove internal fouling (Bouhabila et al. 2001 ; Psoch and Schiewer 2006) . Air sparging is very commonly applied, especially in submerged aerobic MBRs with ultrafiltration and microfiltration membranes, where aeration serves a dual purpose of providing oxygen to bacteria an d mitigating membrane fouling. Coarse air bubbles create shear at membrane surfaces, and partially remove loosely attached 133 fouling layers. It has been well documented that air sparging can enhance hydraulic permeability of MBR membranes with strong positiv e correlations found between air sparging rate and fouling reduction (Chang and Judd 2002 ; Yu et al. 2003 ; Ghosh 2006 ; Fan and Zhou 2007 ; Delgado et al. 2008) . To further reduce membrane fouling, air sparging is of ten coupled with pressure relaxation. (Hong et al. 2002) clearly demonstrated that permeate flux decreased slower when periodical pressure relaxation was applied. (Wu et al. 2008) reached a qualitatively similar conclusion reporting that the extent of fouling was related to the duration and frequency of pressure relaxation. M embrane backwash is another method that is widely used to reduce membrane fouling in MBRs. (Hwang et al. 2009) suggested that backwash by deionized water can completely remove membrane cake and alleviate intrapore fou ling. (Yigit et al. 2009) reported that membrane resistance was reduced ~160% after backwash and concluded that backwash effectively diminish ed reversible fouling due to pore blocking and cake layer formation. Backwash parameters such as duration, interval and backwash flow rate can significantly affect fouling (Wu et al. 2008 ; Hwang et al. 2009) . (Delgado et al. 2008) reported that backwash time had a strong impact on residual fouling. (Kim and DiGiano 2006) showed that higher backwash frequency could reduce long - term fouling rate. With the same backwash volume, higher backwash flux was more effective in fouling reduction than a longer duration of the backwash ( Zsirai et al. 2012) . Enteric viruses, as a type of infectious pathogens in wastewater, pose a significant threat to public safety. Most published studies on virus removal by MBRs focused on bench - and pilot - scale MBR systems and bacteriophages such as MS2 , T4 and F - specific and somatic coliphage (Cicek et al. 1998 ; Hu et al. 2003 ; Shang et al. 2005 ; Comerton et al. 2005 ; Fiksdal and Leiknes 2006 ; Lv et al. 2006 ; Zhang and Farahbakhsh 2007; Zheng and Liu 2007 ; Tam et al. 2007 ; Ravindran et al. 2009 ; 134 Hirani et al. 2010) . Two bench - scale studies employed human viruses; (Madaeni et al. 1995) reported that the removal of poliovirus ranged from 1.3 to 1.8 logs while (Ottoson et al. 2006) showed that the log reduction value (L RV) for enterovirus and norovirus ranged from 0.5 to 1.8 logs. To our knowledge, there have been only five studies on the removal of viruses in full - scale systems. Norovirus removal in a full - scale MBR utilities was reported to cover a very wide range from 0 (i. e. no removal) to 5.5 logs (da Silva et al. 2007) LRVs of ~ 5.1 logs for enteroviruses, 3.9 logs for norovirus, and 5.5 logs for adenoviruses were reported (Kuo et al. 2010 ; Simm ons et al. 2011 ; Simmons and Xagoraraki 2011) . (Zanetti et al. 2010) measured LRVs for F - specific coliphage and somatic coliphage to be 6 logs and 4 logs, respectively. The role of biofilm in virus removal by MBRs has been studied by (Wu et al. 2010) who found that the clean membrane ( = 0.4 µm) contributed only ~0.5 logs removal of somatic coliphages; in contrast, when covered with a biofilm the same membrane could remove 1.8 to 2.6 logs of the virus. Similarly, (Shang et al. 2005) observed that an MBR with the nominal pore size of 0.4 µm could initially (i.e. prior to significant membrane fouling) only remove 0.4 logs of MS - 2 coliphage. After 21 days of operation, the removal efficiency increased to 2.3 logs; it was concluded that membrane biofilm played an important role i n removing the virus. Despite the fact that one or several fouling mitigation methods are routinely applied in MBR plants, little is known about the impact that these practices have on virus removal (Table 4 - 1). Most of the published work on the subject fo cused on chemical cleaning and employed bacteriophages. It has been reported that chemical cleaning that completely removed the membrane biofilm greatly affected the removal of viruses and it could take more than 24 h for the removal to recover to pre - clea ning levels (Lv et al. 2006; Tam et al. 2007) . 135 Only two studies (Lv et al. 2006; Zheng et al. 2005) evaluated the effect of hydraulic flushing (not backwash) by cleaning the membrane surface with tap water, using the same bench scale MBR system and T4 coliphage. To our knowledge, the impacts of pressure relaxation, air scouring and permeate backwash on virus removal in MBR systems have not been investigated yet. The effect of these fouling mitigation methods on the removal of human adenov irus 40 (HAdV 40), an infectious enteric virus is at the focus on the present work. 4. 2. Materials and Methods 4. 2.1. Cell culture experiment and virus incubation A549 cell line has been suggested as an efficient cell line for HAdV (Witt and Bousquet 1988; Lee et al. 2004) and it was used to grow HAdV in this study. Details of virus incubation were described in (Yin et al. 2015) . 4. 2.2. Membrane preparation The polyvinylidene fluoride (PVDF ) hollow fiber membrane used in this work had the nominal pore size of 0.45 µm and the outer diameter of 1.3 mm. Membrane units were made by looping and potting 14 hollow fiber segments (90 cm long each) PFTE tubing wi th an adhesive (Loctite). Each membrane unit had an effective surface area of ~1600 cm 2 , and 4 such units were used in each experiment. Prior to each test, membrane was soaked in deionized (DI) water for at least 24 h, and then compacted by filtering DI wa ter for 12 h. 136 4. 2.3. Bench - scale submerged MBR A schematic of the bench scale MBR system is shown in Figure 4 - 1. The MBR could accommodate 25 L of activated sludge and the working volume was 20 L. A peristaltic digital pump (model 07523 - 80, MasterFlex L/ S) served as the permeate pump. The system was running in a constant flux regime ( Q = 31.3 mL/min; j = 3.26·10 - 6 m/s). Transmembrane pressure ( ) and permeate flow rate were measured by a digital pressure sensor ( Cole - Parmer, 68075 - 00) and digi tal flow meter (model 106 - 4 - C - T4 - C10, McMillan), respectively. A LabView program was developed to (1) maintain the constant permeate flow using a proportional - integral - derivative (PID) algorithm; (2) conduct periodical pressure relaxation by turning the pe rmeate pump on and off; (3) record data from the flow meter and the pressure sensor. Activated sludge from East Lansing wastewater treatment plant was incubated in a 25 L glass cylinder tank with synthetic wastewater (Table 4 - 2) for over three months. Memb ranes were then placed in the activated sludge and the MBR system was run for over three months. The hydraulic retention time (HRT) was 0.5 day, and mixed liquor suspended solids (MLSS) concentration was kept at 4.5 g/L based on daily MLSS measurements. Ae ration was continuously applied throughout the experiment at the rate of 0.57 m 3 /h. A preliminary test indicated that the MBR was able to remove ~97% of total organic carbon (data not shown). 4. 2.4. Fouling and backwash experiments A total of three virus removal experiments were conducted. Each experiment consisted of a 2 - hour water filtration stage (conditioning stage), an 8 - day fouling stage (Stage 1) and two 2 - day backwash stages (Stage 2 and 3). Periodical pressure relaxation was applied during fouling and backwash stages with the formats described 137 in Table 4 - 3. All samples were stored at - 80 °C until analysis. Conditioning stage (duration = 2 h): A set of pristine membranes was placed in a tank with 20 L of DI water and air diffuser on the bottom. Vi rus was added into the tank and mixed for 1 h by aeration. Membrane filtration was carried out at a constant flow rate of 31.3 mL/min for 1 h. Feed and permeate samples were collected at the end of the stage. Stage 1 (duration = 8 d): The membrane was inst alled in the MBR system and was then operated as described in S ection 4. 2.3 . Two sets of feed and permeate samples were collected each day. For sampling, 40 mL of virus stock solution was spiked into the activated sludge ~ 6 min before pressure relaxation . The first set of samples was taken 50 s before pressure relaxation, while the second set of samples was collected 3 min (when flow rate was constant) after pressure relaxation. Feed samples with activated sludge were settled for 15 min, and then passed t hrough 0.22 µm Millipore filters. The virus concentration in the filtrate (assumed to represent the liquid phase of the mixed liquor) was considered as the feed concentration of the virus. Stages 2 and 3 (duration = 2 d each): At the beginning of each of these stages, the membrane was backwashed using permeate water following the format described in Table 4 - 3 . Two sets of samples were collected every 16 h, following the same sampling strategy as in Stage 1. 4. 2.5. DNA extraction and quantitative polymeras e chain reaction (qPCR) Virus DNA in each sample was extracted by using MagNa Pure Compact System with Nucleic Acid Isolation Kits (Roche Applied Sciences) , following the 138 recov ery. The DNA extracts were stored at - 80 °C immediately after extraction. Following DNA extraction, virus quantification was conducted using qPCR ( Roche Light Cycler). Sequences of primers and TaqMan probe were adopted from (Heim et al. 2003) . The sequ ence (5' - 3') of forward primer, reverse primer and probe are GCCACGGTGGGGTTTCTAAACTT, GCCCCAGTGGTCTTACATGCACATC, and FAM - TGCACCAGACCCGGGCTCAGGTACTCCGA - TAMRA, respectively. HAdV concentration was calculated based on crossing point C p values generated by Lig htCycler software and a previously developed standard curve. 4. 2.6. Inhibition test of qPCR Polymerase chain reaction may be inhibited in the presence of organic matter . In order to rule out the potential inhibition on qPCR by organic matter in the activated sludge, an inhibition test was conducted: 1 mL of HAdV stock solution was added into 9 mL of DI water and 9 mL of feed sample. DNA extraction and qPCR were carried out accordingly. No significant difference of measured virus concentration between DI water and feed sample was found. 4. 3. Results and Discussion 4. 3.1. Membrane fouling and transmembrane pressure buildup Figure 4 - 2 summarizes profiles in all three experiments. The duration of the filtration period in each cycle in exp. 1 and exp. 2 was 25 min, while in exp. 3 it was 50 min (Table 4 - 3). The LabView program was used to log in data every second. Each dot in Figure 4 - 2 represents the average value of from the 3 rd min (when the flow rate becomes constant) to the end of filtration period in each cycle. In exp. 1 and exp. 2, which were performed in the 25 min/5 min filtration/relaxation cycles, 139 increased from ~ 3.6 kPa to 11 kPa during 8 days of the fouling test. In exp. 3 (performed in 50 min /10 min filtration relaxation cycles) increased from ~ 4.0 kPa to 15 kPa over the same period. The results suggest that less freque nt cycling leads to more fouling even though the ratio of relaxation time ( ) to filtration time ( is maintained the same. One explanation for this trend is that fouling accumulates over the entire filtration stage of the cycle whi le the capability of air sparging to remove fouling during the relaxation stage is limited so that only the most recently (less than 50 min in our experimental conditions) formed layer can be removed by aeration. Similar results were reported by (Wen et al. 1999) , with m ore fouling observed in an MBR with an operational mode of 8 min filtration / 2 min relaxation compared to 4 min filtration/ 1 min relaxation. They also observed that the 2 min/ 0.5 min format resulted in more fouling than the 4 min on/ 1 min off format. W u et al. (2008) found the 220 s / 20 s off format created more fouling than the 440 s / 40 s. This is probably because of 20 to 30 s is insufficient for air sparging to remove all reversible fouling. The data also show how the backwash flow ra te and duration affect . In exp.1, backwash was conducted at 40 mL/min rate for 10 min, and the decreased by 3.6 kPa and 2.5 kPa in Stage 2 and 3, respectively. In exp. 2 with 20 min backwash at 20 mL/min rate, the decreased by 2.6 kPa and 1.6 kPa in Stage 2 and 3, respectively. With the same backwash flow rate and duration, in exp. 3 dropped by 3.5 kPa at Stage 2, which is similar to what was observed in exp. 1. However, only decreased by 1.0 kPa in Stage 3. These data indicate that with a given backwash volume, backwash flux is more effective than backwash duration in controlling . This is consistent with results reported by (Z sirai et al. 2012) , who made a similar conclusion based on their results with a pilot - scale submerged MBR. Moreover, the 140 data also show that the effect of backwash on was weaker when the membrane was subject to the 2 nd backwash compared to t he 1 st backwash, and this tendency seems to be enhanced with a longer duration of the filtration/relaxation cycle (exp. 3). 4. 3.2. Virus removal The removal of HAdV 40 from DI water in experiments 1, 2 and 3 was 1.22, 1.07 and 1.07 logs respectively. When the membrane units were placed in activated sludge and the filtration was conducted at the same flow rate, LRV increased to ~ 2 logs in all three experiments ( Figure 4 - 3). This conflicts with the data presented by (Shang et al. 2005) where the initial LRV from activated sludge was almost the same ( ~ 0.3 log) as the removal from DI water. This is because the first sample in our experiments was collected when filtration had been carried out for ~ 25 min (exp. 1 and exp. 2) or 50 min (exp. 3), and membrane fouling occurred during that time. More import antly, even though the membrane used by (Shan g et al. 2005) had a pore size (0.4 µm) similar to that of the membrane employed in this work, the virus used in their study (MS - 2 phage) was much smaller (20 - 25 nm, (Shang et al. 2005) ) than the human adenovirus 40 (70 - 140 nm, (Xagoraraki et al. 2014) ) . The p ore blockage effect on the removal of MS - 2 phage is not as significant as on the removal of HAdV 40, since it is easier for smaller viruses to pass through partially blocked pores. In exp. 1, LRV increased from 2.33 logs (before relaxation) and 2.06 logs ( after relaxation) at the beginning, to 3.87 logs and 2.78 logs at day 4 respectively. Then LRV remained at the approximately same level for the last 4 days. In exp. 2, the observed LRV started at ~2 logs, increased to 4.19 logs (before relaxation) and 3.54 logs (after relaxation) at day 4, and ended at 4.70 logs and 3.67 logs at day 8. Thus virus removal increased much faster during the first 4 days compared to last 4 days. 141 A similar trend was observed in previous studies. In the 20 - day experiment carried o ut by (Shang et al. 2005) , the LRV of MS - 2 g rew from 0.3 logs to 2.3 logs in the first 10 days, then reached 2.5 logs at the 20 th day. (Madaeni et al. 1995) implemented a 6 - h experiment with 0.22 µm PVDF membranes, in which the increase of poliovirus rejection was rapid between 0.5 h a nd 2 h, and slowed down afterward. Such removal profile has also been reported for chemicals. In a 35 - h experiment with a pilot - scale side - stream MBR, the removals of nitrate, total organic carbon and alachlor sharply increased in the first 5 10 hours an d then remained relatively constant during the rest of the experiment (Ravindran et al. 2009) . Virus removal in exp. 3 in our study increased steadily throughout Stage 1. A larger number of samples taken closer to the end of Stage 1 wo uld be needed to further investigate the trend. In experiment 1 and 3, a backwash was applied for 10 min at the flow rate of 40 mL/min prior to each stage 2 and stage 3. In exp. 1, the LRV before and after pressure relaxation decreased by 0.91 and 0.72 lo g at the result of the first backwash and by 0.87 and 0.60 log as a result of the second backwash, respectively. In exp. 3, LRV reduced by 0.76 and 0.87 log due to the 1 st backwash, while the difference between LRVs before and after the 2 nd backwash could not be calculated as virus concentrations in permeate samples at day 10 in Stage 3 were below the detection limit. The same volume of membrane permeate was used in exp. 2 for backwash, but at the flow rate of 20 mL/min for 20 minutes. As a result, backwash 1 lowered LRV by 0.33 and 0.31 log while backwash 2 barely affected the LRV. Reduction of virus removal caused by different backwash formats is summarized in Figure 4 - 4, and t - test shows that LRV reduction due to backwash with higher flow rate is signifi cantly greater (p < 0.05) than with a longer duration backwash. These data demonstrate that backwash had a similar impact on and virus removal: (1) with the same permeate volume used for backwash, higher backwash flux causes a 142 larger reductio n LRV than a longer backwash does; (2) Decrease of virus removal appears to be greater during the 1 st backwash compared to the 2 nd backwash. (Wu et al. 2010) studied the impact of chemical backwash (by a NaClO solution) on virus removal in a full - scale MBR. They found virus rejection by membrane dropped 0 1.5 logs after each backwash. In all our experiments, the virus removal recovered to the pre - backwash level within 16 h after the backwash was applied. In contrast, (Tam et al. 2007) observed that it may take more than 24 h for the recovery of virus removal after the fouled membrane is subjected to chemical cleaning. As shown above, longer filtration/relaxation cycles cause d higher . However, relaxation nor after relaxation in Stage 1 of experiment 3 is significantly higher (p > 0.05) compared to experiment 1 and 2 combined. Moreover, it is notable that in all sampling events, virus removal before pressure relaxation is always higher than after pressure relaxation, and the mean LRV before relaxation was 0.74, 0.48, and 0.42 log higher in Stage 1 of experiment 1, 2 and 3, respe ctively. This suggests that the portion of fouling that can be reversed by aeration during pressure relaxation can enhance virus removal. Our previous study indicates that the reversible fouling caused by silica particles (3.45 µm in diameter) reduced viru s removal, especially in the case small pore size membranes (Yin et al. 2015) . This suggests that reversible fouling could either increase or decrease virus removal, and that the property of foul ants is the dominant factor in this regard. As shown in Figure 4 - 5, this enhancement in LRV due to reversible fouling appears to be unaffected by the duration of filtration/relaxation cycles, as there was no significant difference (p > 0.05) between combin ed data from exp. 1 and exp. 2 on the one hand and the data from exp. 3 on the other hand. This is probably because over the short term, increase is caused by reversible fouling. In each filtration cycle 143 after the flow rate reached a constant level, the change in was very slow. This is supported by the t - test which showed the difference of before and after pressure relaxation at sampling events in exp. 3 was not significantly (p > 0.05) higher compared to exp.1 and exp. 2 combined. Thus did not increase further in each filtration cycle despite the fact that duration of filtration cycles was doubled. 4. 3.3. Relationship between transmembrane pressure and virus removal Figure 4 - 6 demonstrates the correla tion between transmembrane pressure ( and virus removal (LRV). and LRV in exp. 1 and exp. 2 were moderately correlated ( R 2 = 0.63 and 0.78, respectively) to each other, while a relatively strong correlation ( R 2 = 0.94) was observ ed in exp. 3. A moderate correlation ( R 2 = 0.72) was obtained when the analysis was applied to data from all three experiments. The correlations found in our study are stronger than those observed by (Wu et al. 2010) , who reported a moderate correlation ( R 2 = 0.656) be tween and LRV of indigenous somatic coliphages. The possible correlation between and LRV was also explored by (Shang et al. 2005) , but these authors suggested that the correlation only exists when the food to mass ratio is low. In sum, may be used to estimat e levels of virus removal in MBR systems, and higher generally leads to greater virus removal. However, the quantitative correlations may be system - dependent and vary with the virus type. 4. 4. Conclusion s This study demonstrated the change o f transmembrane pressure (TMP) and removal of human adenovirus, when periodical pressure relaxation and permeate 144 backwash were applied in a bench - scale MBR. Based on the data presented above, following conclusions can be drawn: Both pressure relaxation and permeate backwash can mitigate membrane fouling, and meanwhile decrease virus removal. Reversible fouling plays an important role in removing viruses in MBRs. With same permeate volume for backwash, higher backwash flux can cause more reduction in TMP and virus removal. With same filtration/relaxation ratio, longer cycle will lead to higher extent of fouling, but its impact on virus removal is not significant. TMP may be used to estimate level of virus removal in MBRs. Higher TMP generally leads to greate r virus removal. But the quantitative correlation between TMP and virus removal is not very persistent. 145 APPENDIX 146 Table 4 - 1. Effect of membrane fouling mitigation methods on virus removal in submerged MBRs MBR Fouling mitigation method Effec t on fouling Virus Effect on virus removal Reference Bench scale 0 .4 µm Chemical backwash NA Somatic coliphage * Wu et al. 2010 0.4 µm Chemical backwash Original pressure recovered MS - 2 coliphage 0.4 < Shang et al. 2005 Pilot scale 0.4 µm Chemical clean NA F - specific coliph age Concentration in the effluent increased from 0.5 to 18.5 PFU/100 mL Tam et al. 2007 0.1 µm Chemical clean NA MS - 2 coliphage Concentration in the effluent increased by up to 32 PFU/100 mL Hirani et al. 2014 Bench scale, 0.1 µm and 0.22 µm Tap water flush Hydraulic resistance decreased from 12 12 m - 1 T4 coliphage Concentration in the effluent increas ed from <2 to 400 - 500 PFU/mL Zheng et al. 2005 chemical clean Hydraulic resistance further decreased 12 12 m - 1 Concentration in the effluent further increased to 2000 - 16000 PFU/mL 0.1 µm Tap water flush Hydraulic resistance decreased from 13 12 m - 1 T4 coliphage LRV decreased fr om 6 logs to 5 logs Lv et al. 2006 chemical clean Hydraulic resistance further decreased 12 m - 1 LRV further decreased to 4 logs 0.22 µm Tap water flush Hydraulic resistance decreased from 12 12 m - 1 LRV decreased from 5 logs to 3.3 logs chemical clean Hydraulic resistance further decreased 12 m - 1 LRV further decreased to 1.9 logs 147 Table 4 - 2. Composition of the synthetic wastewater * Chemicals Daily dose (g) Chemicals Daily dose (mg) Glucose 15 H 3 BO 3 3.60 Peptone 5 CuSO 4 · 5H 2 O 0.72 KH 2 PO 4 1.6 KI 5.12 (NH 4 ) 2 SO 4 5.2 MnCl 2 · 4H 2 O 2.88 MgSO 4 · 7H 2 O 3.2 NaMoO 4 · 2H 2 O 1.44 CaCl 2 · 2H 2 O 1.6 ZnSO 4 · 7H 2 O 2.88 EDTA 0.24 CoCl 2 · 6H 2 O 3.60 NaCl 5 FeCl 3 · 6H 2 O 36.00 * The pH of the synthetic wastewater was adjusted to 7.5 before use. The mineral makeup of the synthetic wastewater was adapted from (Yuan et al. 2009) and (Broughton et al. 2008) . 148 Table 4 - 3. Parameters of pressure relaxation and backwash Exp. #. Pressure relaxation ( Permeate backwash ( Flow rate 1 25 min / 5 min 40 mL/min 10 min 2 25 min / 5min 20 mL/min 20 min 3 50 min / 10 min 40 mL/min 10 min 149 Figure 4 - 1. Schematic of the submerged MBR 150 Figure 4 - 2. Transmembrane pressure as a function of filtration time and the effect of backwash 151 A B Figure 4 - 3. Effects of pressure relaxation and backwash on the removal of HAdV 40 in submerged MBR operated under three different filtration / pressure relaxation sc hedules and backwash protocols : A: exp. 1; B: exp. 2; C: exp. 3 152 Figure 4 - 3. C 153 Figure 4 - 4. Decrease in virus removal as a result of backwash for two different backwash formats 154 Figure 4 - 5 . Decrease in virus removal as a result of pressure relaxation for two different formats of the filtration/relaxation (F/R) cycle. 155 A B Figure 4 - 6. Correlations between virus removal and transmembrane pressure in exp eriments 1 (A), 2 (B) and 3 (C)* * For each data point, represents the pressure averaged over 11 s interval around the corresponding sampling point 156 Figure 4 - 6. C 157 REFERENCES 158 R EFERENCES Beaubien, A., Baty, M., Jeannot, F., Francoeur, E., & Manem, J. (1996). Design and operation of anaerobic membrane bioreactors: development of a filtration testing strategy. Journal of Membrane Science , 109 (2), 173 - 184. Bouhabil , R. B., Buisson, H. (2001). Fouling characterisation in membrane bioreactors. Separation and Purification Technology , 2 2 , 123 - 132. Broughton, A., Pratt, S., & Shilton, A. (2008). Enhanced biological phosphorus removal for high - strength waste water with a low rbCOD: P ratio. Bioresource Technology , 99 (5), 1236 - 1241. Chang, I. S., Le Clech, P., Jefferson, B., & Judd, S. (2002). Membrane fouling in membrane bioreactors for wastewater treatment. Journal of E nvironmental E ngineering , 128 (11), 1018 - 1029. Chang, I. S., & Judd, S. J. (2002). Air sparging of a submerged MBR for municipal wastewater treatment. Process Biochemistry , 37 (8), 915 - 920. Choi, J. G., Bae, T. H., Kim, J. H., Tak, T. M., & Randall, A. A. (2002). The behavior of membrane fouling i nitiation on the crossflow membrane bioreactor system. Journal of Membrane Sci ence , 203 (1), 103 - 113. Cicek, N., Winnen, H., Suidan, M. T., Wrenn, B. E., Urbain, V., & Manem, J. (1998). Effectiveness of the membrane bioreactor in the biodegradation of high molecular weight compounds. Water Research , 32 (5), 1553 - 1563. Comerton, A. M., Andrews, R. C., & Bagley, D. M. (2005). Evaluation of an MBR RO system to produce high quality reuse water: Microbial control, DBP formation and nitrate. Water R esearch , 39 (16), 3982 - 3990. Cornel, P., & Krause, S. (2008). Membrane bioreactors for wastewater treatment. Advanced Membrane Technology and Applications , 217 - 238. da Silva, A. K., Le Saux, J. C., Parnaudeau, S., Pommepuy, M., Elimelech, M., & Le Guyader, F. S. (2007). Ev aluation of removal of noroviruses during wastewater treatment, using real - time reverse transcription - PCR: different behaviors of genogroups I and II. Applied and Environmental Microbiology , 73 (24), 7891 - 7897. Delgado, S., Villarroel, R., & González, E. (2 008). Effect of the shear intensity on fouling in submerged membrane bioreactor for wastewater treatment. Journal of Membrane Science , 311 (1), 173 - 181. Fan, F., & Zhou, H. (2007). Interrelated effects of aeration and mixed liquor fractions on membrane foul ing for submerged membrane bioreactor processes in wastewater treatment. Environmental S cience & T echnology , 41 (7), 2523 - 2528. Fiksdal, L., & Leiknes, T. (2006). The effect of coagulation with MF/UF membrane filtration for the removal of virus in drinking water. Journal of Membrane 159 Science , 279 (1), 364 - 371. Ghosh, R. (2006). Enhancement of membrane permeability by gas - sparging in submerged hollow fibre ultrafiltration of macromolecular solutions: role of module design. Journal of M embrane S cience , 274 (1), 7 3 - 82. Ghosh, R. (2006). Enhancement of membrane permeability by gas - sparging in submerged hollow fibre ultrafiltration of macromolecular solutions: role of module design. Journal of Membrane Science , 274 (1), 73 - 82. Gujer, W., Henze, M., Mino, T., & van Loo sdrecht, M. (1999). Activated sludge model no. 3. Water Science and Technology , 39 (1), 183 - 193. Heim A., Ebnet C., Harste G., Pring - Åkerblom P. (2003). Rapid and Quantitative Detection of Human Adenovirus DNA by Real - Time PCR. Journal of Medical Virology, 70(2): 228 239 Hirani, Z. M., DeCarolis, J. F., Adham, S. S., & Jacangelo, J. G. (2010). Peak flux performance and microbial removal by selected membrane bioreactor systems. Water R esearch , 44 (8), 2431 - 2440. Hirani, Z. M., Bukhari, Z., Oppenheimer, J., J jemba, P., LeChevallier, M. W., & Jacangelo, J. G. (2014). Impact of MBR cleaning and breaching on passage of selected microorganisms and subsequent inactivation by free chlorine. Water R esearch , 57 , 313 - 324. Hu, J., Ong, S., Song, L., Feng, Y., Liu, W., T an, T., Lee, L., & Ng, W. (2003). Removal of MS2 bacteriophage using membrane technologies. Water Sci ence and Technol ogy , 47 (12), 163 - 168. Hwang, K. J., Chan, C. S., & Tung, K. L. (2009). Effect of backwash on the performance of submerged membrane filtrati on. Journal of Membrane Science , 330 (1), 349 - 356. Judd, S. (2008). The status of membrane bioreactor technology. Trends in biotechnology , 26 (2), 109 - 116. Judd, S., (2010). The MBR book: P rinciples and A pplications of M embrane B ioreactors for W ater and W ast ewater T reatment . Elsevier. Kim, J., & DiGiano, F. A. (2006). A two - fiber, bench - scale test of ultrafiltration (UF) for investigation of fouling rate and characteristics. Journal of M embrane S cience , 271 (1), 196 - 204. Kishino, H., Ishida, H., Iwabu, H., & N akano, I. (1996). Domestic wastewater reuse using a submerged membrane bioreactor. Desalination , 106 (1), 115 - 119. Kuo, D. H. W., Simmons, F. J., Blair, S., Hart, E., Rose, J. B., & Xagoraraki, I. (2010). Assessment of human adenovirus removal in a full - sca le membrane bioreactor treating municipal wastewater. Water R esearch , 44 (5), 1520 - 1530. Lee, C., Lee, S. H., Han, E., & Kim, S. J. (2004). Use of cell culture - PCR assay based on combination of A549 and BGMK cell lines and molecular identification as a 160 tool to monitor infectious adenoviruses and enteroviruses in river water. Applied and E nvironmental M icrobiology , 70 (11), 6695 - 6705. Lv, W., Zheng, X., Yang, M., Zhang, Y., Liu, Y., & Liu, J. (2006). Virus removal performance and mechanism of a submerged membr ane bioreactor. Process Biochemistry , 41 (2), 299 - 304. Madaeni, S. S., Fane, A. G., & Grohmann, G. S. (1995). Virus removal from water and wastewater using membranes. Journal of Membrane Science , 102 , 65 - 75. Ottoson, J., Hansen, A., Björlenius, B., Norder, H., & Stenström, T. A. (2006). Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant. Water Research , 40 (7), 1449 - 1457. Pankhania, M., Stephenson, T., & Semmens, M. J. (1994). Hol low fibre bioreactor for wastewater treatment using bubbleless membrane aeration. Water R esearch , 28 (10), 2233 - 2236. Psoch, C., & Schiewer, S. (2006). Anti - fouling application of air sparging and backflushing for MBR. Journal of Membrane Science , 283 (1), 27 3 - 280. Ravindran, V., Tsai, H. H., Williams, M. D., & Pirbazari, M. (2009). Hybrid membrane bioreactor technology for small water treatment utilities: process evaluation and primordial considerations. Journal of Membrane Science , 344 (1), 39 - 54. Shang, C., Wong, H. M., & Chen, G. (2005). Bacteriophage MS - 2 removal by submerged membrane bioreactor. Water R esearch , 39 (17), 4211 - 4219. Simmons, F. J., Kuo, D. H. W., & Xagoraraki, I. (2011). Removal of human enteric viruses by a full - scale membrane bioreactor dur ing municipal wastewater processing. Water R esearch , 45 (9), 2739 - 2750. Simmons, F. J., & Xagoraraki, I. (2011). Release of infectious human enteric viruses by full - scale wastewater utilities. Water Research , 45 (12), 3590 - 3598. Sutloviæ, D., Gojanoviæ, M. D Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid. Humic Acid , 2 , 18 - 21. Tam, L. S., Tang, T. W., Lau, G. N., Sharma, K. R., & Chen, G. H. (2007). A pi lot study for wastewater reclamation and reuse with MBR/RO and MF/RO systems. Desalination , 202 (1), 106 - 113. Vaid, A., Kopp, C., Johnson, W., & Fane, A. G. (1991). Integrated waste water treatment by coupled bioreactor and membrane system. Desalination , 83 (1), 137 - 143. Van der Roest, H. F., Lawrence, D. P., & Van Bentem, A. G. N. (2005). Membrane bioreactors for municipal wastewater treatment. IWA Publishing. Wen, C., Huang, X., & Qian, Y. (1999). Domestic wastewater treatment using an 161 anaerobic bioreactor coupled with membrane filtration. Process Biochemistry , 35 (3), 335 - 340. Witt, D. J., & Bousquet, E. B. (1988). Comparison of enteric adenovirus infection in various human cell lines. Journal of V irological M ethods , 20 (4), 295 - 308. Wu, J., Le - Clech, P., Stu etz, R. M., Fane, A. G., & Chen, V. (2008). Effects of relaxation and backwashing conditions on fouling in membrane bioreactor. Journal of Membrane Science , 324 (1), 26 - 32. Wu, J., Li, H., & Huang, X. (2010). Indigenous somatic coliphage removal from a real municipal wastewater by a submerged membrane bioreactor. Water R esearch , 44 (6), 1853 - 1862. Xagoraraki, I., Yin, Z., & Svambayev, Z. (2014). Fate of viruses in water systems. Journal of Environmental Engineering , 140 (7), 04014020. Xie, K., Lin, H. J., Mahe ndran, B., Bagley, D. M., Leung, K. T., Liss, S. N., & Liao, B. Q. (2010). Performance and fouling characteristics of a submerged anaerobic membrane bioreactor for kraft evaporator condensate treatment. Environmental T echnology , 31 (5), 511 - 521. Yigit, N.O. , Civelekoglu, G., Harman, I., Koseoglu, H., Kitis, M. (2009). Effects of various backwash scenarios on membrane fouling in a membrane bioreactor. Desalination, 237(1 - 3), 346 - 356. Yin, Z., & Xagoraraki, I. (2014). Membrane Bioreactors (MBRs) for Water Reus e in the USA. The Handbook of Environmental Chemistry 2015. Series ISSN: 1867 - 979X. Springer Berlin Heidelberg . Yin, Z., Tarabara, V. V., & Xagoraraki, I. (2015). Human adenovirus removal by hollow fiber membranes: Effect of membrane fouling by suspended a nd dissolved matter. Journal of Membrane Science , 482 , 120 - 127. Yu, K., Wen, X., Bu, Q., & Xia, H. (2003). Critical flux enhancements with air sparging in axial hollow fibers cross - flow microfiltration of biologically treated wastewater. Journal of Membran e Science , 224 (1), 69 - 79. Yuan, Q., Sparling, R., & Oleszkiewicz, J. A. (2009). Waste activated sludge fermentation: effect of solids retention time and biomass concentration. Water Research, 43 (20), 5180 - 5186. Zanetti, F., De Luca, G., & Sacchetti, R. (20 10). Performance of a full - scale membrane bioreactor system in treating municipal wastewater for reuse purposes. Bioresource Technology , 101 (10), 3768 - 3771. Zhang, K., & Farahbakhsh, K. (2007). Removal of native coliphages and coliform bacteria from munici pal wastewater by various wastewater treatment processes: implications to water reuse. Water R esearch , 41 (12), 2816 - 2824. Zheng, X., Lv, W., Yang, M., Liu, J., 2005. Evaluation of virus removal in MBR using coliphage T4. Chinese Science Bulletin 2005 , 50 ( 9 ) , 862 - 867. 162 Zheng, X., & Liu, J. (2007). Virus rejection with two model human enteric viruses in membrane bioreactor system. Science in China Series B: Chemistry , 50 (3), 397 - 404. Zsirai, T., Buzatu, P., Aerts, P., & Judd, S. (2012). Efficacy of relaxation, backflushing, chemical cleaning and clogging removal for an immersed hollow fibre membrane bioreactor. Water R esearch , 46 (14), 4499 - 4507. 163 CHAPTER 5 ADSORPTION AND DESORPTION OF HUMAN ADENOVIRUS TO PRIMARY AND SECONDARY SLUDGE Abstract The presence of human enteric viruses in water, and their resulting potential to cause diseases, posed a threat on public health. Virus adsorption to sludge particles has been suggested as one of the major mechanisms of virus removal, while the studies focused on sorptio n kinetics of viruses in sludges are limited. With assistance of real - time quantitative polymerase chain reaction (qPCR), we explored the adsorption and desorption of human adenovirus 40 (HAdV) in primary and secondary sludge. The results showed that great er HAdV adsorption was observed when sludge filtrate was used as solute compared to DI water. Adsorption of HAdV conformed to Freundlich isotherm, and it exhibited very similar behavior in the two types of sludge. Desorption of HAdV from sludge particles w as not very significant in sequential desorption experiments. More HAdV was desorbed from primary sludge than from secondary sludge, but the difference was not statistically significant. Key words: adsorption, desorption, human adenovirus, Freundlich iso therm 164 5. 1. Introduction 5. 1.1. Viruses in the wastewater Enteric viruses pose a considerable threat to human health due to their low infectious dose and long survival in the environment. V iruses have been noted in the contaminant candidate lists (CCL) is sued by U.S. Environmental Protection Agency (USEPA) , including adenovirus, enterovirus, coxsackievirus , echovirus, hepatitis A virus, and calicivirus (Xagoraraki et al. 2014). A large number of enteric viruses are excreted in human feces and urine, which makes wastewater one of the most concentrated sources of viruses (Puig et al . 1994, Castignolles et al . 1998). It has been reported that virus concentration in wastewater could be up to 10 9 copies per liter (da Silva et al . 2007; Kuo et al. 2010; Simmons e t al. 2011). Therefore, it is critical to remove viruses from wastewater before discharging it into the en vironment. However, wastewater treatment systems may not be able to serve as an absolute barrier against contaminants, and the presence of enteric vir uses are frequently reported in treated wastewater, even in the effluent from membrane bioreactors (MBR), the most advanced wastewater treatment systems (Xagoraraki et al. 2014; Simmons et al. 2011; Kuo et al. 2010). Wastewater effluents have been consider ed as one of major potential sources of pathogens (Bitton and Harvey 1992). Sludge, containing viruses, from wastewater treatment process is likely to be applied to landfill s , and biosolids (treated sludge) containing viruses, may be applied on agricultura l land (Wong et al. 2010 ). 5. 1.2. Virus sorption mechanisms Transport and survival of viruses in the environment is largely controlled by adsorption and desorption. Conventional activated sludge is the most widely used wastewater treatment system worldwi de, where adsorption to biosolids is the one of 165 the major mechanisms for virus removal (Gerba et al. 1975; Vilker et al. 1980; Gerba 1984; Kim and Uno 1996). Rainfall may cause virus desorption from land applied biosolids and contaminate groundwater, which is one of the major sources of drinking water. When viruses come to the surface water syste ms, adsorption to suspended particles may facilitate virus survival and transport, while soil or sediment can serve as a reservoir and shade for viruses to survive (Gerba and Schaiberger 1975 ; Hurst et al. 1980) . Consequently, understanding virus adsorption and desorption behavior in different environmental circumstances is a key step to prevent people from exposure to pathogenic viruses. Virus adsorption is type and strain specific ( Boche and Quilligan 1966; Goyal and Gerba 1979; Gerba et al. 1980 ; Gerba et al. 1981 ). L arger virus size facilitates adsorption due to more available surface charges ( Dowd et al., 1998; Chattopadhyay and Puls, 1999 ). Surface charge is an important factor for virus adsorption, and the attractive forces between sorbents and viruses tend to be stronger if they have opposite surface charge. Hydropobicity is widely used to explain different affinity between different types of virus es and sorben ts (Ivanova et al. 2011 ; Han et al. 2006; Bales et al. 1991 ). It has been concluded that hydrophobic sorbents are more favored to adsorb hydrophobic viruses, and vice versa (Chattopadhyay et al. 2002). Additionally , the form ing of hydrogen and/or hydroxyl bond between sorbents and viruses has been suggested as an important factor enhancing adsorption ( Oza and Chaudhuri 1975; Oza and Chaudhuri 1976 ). Water content, also known as moisture content, refers to the amount of water contained in a material. An agre ement appears to be reached among previous literature that virus adsorption is enhanced at lower water content in all kinds of sorbents ( Powelson et al. 1990; Jin et al. 2000; Zhao et al. 2008; Han et al. 2006; Yeager and O'Brien 1979; Poletika et al. 1995 ; Chu et al. 2001 ; Chu et al. 2003). 166 Three mechanisms have been proposed for this phemenemon: (1) adsorption is promoted in the solid - water interface of media with lower water content due to higher extend of proximity between viruses and so lid surface (Pre ston and Farrch 1988; Bitton et al. 1984 ); (2) the air - water interface (AWI), which only exists in unsaturated media, can provide additional sorption sites for colloid particles to attach ( Wan and Wilson 1994; Powelson and Mills 1996; Jewett et al. 1999 ); (3) water content can also influence virus transport by film st raining effect that transport of colloidal particle s is restricted in porous media when the thickness of water film is smaller than the particle diameter ( Wan and Tokunaga 1997 ; Han et al. 2006 ). Greater virus adsorption to sorbents is usually observed at lower pH (Schulze - Makuch et al. 2003; Chaudhuri et al. 1977; Drewry and Eliassen 1968; Zhao et al. 2008; Oza and Chaudhuri 1976; You et al. 2003; Bales et al. 1993 and 1995), but not all sorpti on experiments from previous studies followed the same trend ( Cookson 1969; Oza and Chaudhuri 1975). pH can govern virus sorption by altering the surface charge. For example, soils and viruses are usually negatively charged in natural environment (Oze and Chaudhuri 1976; Bitton 1975). With increased pH, the charge on both virus and the sorbent surfaces becomes more negative, between which the adsorption becomes weaker due to increased electrostatic repulsio n among each other (Zhao et al. 2008; Drewry and El iassen 1968; Chaudhuri et al. 1977 ). The Derjaguin Landau Verwey Overbeek (DLVO) theory, also known as double layer theory, is frequently employed to explain the impact of pH on virus sorption: when pH is increased, the diffused layer becomes thinner, and surfaces of virus es and sorbents have greater opportunities to approach each, where the van der Waals attraction becomes more sign ificant (Gerba 1984). Moreover, tail fibers on virus surface are thought to attach on sorbent, and they are extended for adsor ption at pH between 6.0 and 9.5. Otherwise, they will attach to tail sheath, and the viral 167 adsorption is weakened (Cookson 1969). Higher ionic strength usually leads to greater virus adsorption ( Carlson et al. 1968; Chaudhuri et al. 1977; Drewry and Eliass en 1968; Cao et al. 2010; Bradley et al. 2011; Oza and Chaudhuri 1976; Preston and Farrah 1988; Grant et al. 1993; Lance and Gerba 1984; Lipson and Stotzky 1983; Pham et al. 2009; Wallis and Melnick 1967 ), but not always ( Chu et al. 2000; Zhuang and Jin 20 03a; Cookson 1969; Penrod et al. 1996; Thompson and Y ates 1999; Bales et al. 1993 ). Three mechanisms have been proposed: (1) according to DLVO theory, the electric double layer around viruses and sorbents will be squeezed due to high ionic strength, and th ose colloidal particles have greater chance to get close to each other, which leads to more adsorption (Chu et al. 2000; Lance and Gerba 1984 ; Lance et al. 1976; Bitton 1975); (2) the salt ions in aqueous solutions shield surfaces of viruses and sorbents a nd prevent them from interacting with each other; (3) virus fibers extend for sorption at low ionic strength, and attach to tail sheat h when the ionic strength is high (Cookson 1969). It has been well accepted that organic matter inhibits virus adsorption and enhances desorption ( Carlson et al. 1968; Powelson et al. 1991; Lance and Gerba 1984; Lipson and Stotzky 1984; Stagg et al. 1977; Bixby and O'Brien 1979; Gu ttman - Bass and Catalano - Sherman 1986; Wong et al. 2013; Lo and Sproul 1977; Pham et al. 2009; Sc heuerman et al. 1979; Bales et al. 1993; Cliver 1968; Ryan et al. 1999 ) . Competition between organic material and virus particles for sorption sites appears to be the most popular mechanisms for the inhibition of viral adsorption (Carlson et al. 1968; Powe lson et al . 1991; Zhuang and Jin 2003a; Bixby and O'Bri en 1979; Lo and Sproul 1977; Pieper et al. 1997). It also has been suggested that organic material decreases virus adsorption by modif ying surfaces of viruses and sorbents, or forming inert complex wit h viruses ( Zhuang and Jin 2003a; Bixby and O'Brien 1979). 168 5. 1.3. Virus sorption in activated sludge and biosolids Adsorption to sludge particles has been suggested as the m ajor mechanism of virus removal in wastewater treatment processes (Gerba et al. 197 5; Vilker et al. 1980; Gerba 1984). Virus partitioning and removal due to sorption in activated sludge is summarized in Table 5 - 1. Enteroviruses showed higher adsorptiove affinity to activated sludge than rotaviruses (Farrah et al. 1978). Balluz et al. (19 78) reported that virus (f2 coliphage) distribution in liquid and solid phase was at the ratio of 18:82 in activated sludge. Rao et al. (1987) observed that 92% spiked rotavirus was attached to suspended solids in activated sludge. Similar results were obt ained by Englande et al. (1983), who collected samples from multiple municipal wastewater treatment plants, and demonstrated the majority of viruses (mostly > 90%) were associated with solids. Moore et al. (1978) observed approximate ly 83 - 99% of the indi genous enteroviruses were attached to solids. Virus partition between solid and liquid phase is determined by adsorption capacity of activated sludge (Arraj et al. 2005). Presence of adenoviruses, enteroviruses and noroviruses in dewatered sludge and class B biosolids has been previously reported, of which the concentration could be up to 10 8 copies per gram (Bofill - Mas et al . 2006; Monpoeho et al. 2001 and 2004; Viau and Peccia 2009; Wong et al. 2010). Adsorption is a reversible process, which depends on t emperature, pH, ionic strength, soil properties and virus type and amount (Jørgensen and Lund 1986). Virus detachment from biosolids is of concern because more than half of biosolids generated in the United States are applied to landfill s (NRC 2002). Chang e of environmental conditions, such as rainfall, may enhance desorption of virus and cause contamination (Englande et al. 1983; Landry et al. 1980). Desorption of viruses from sludge/biosolids is present in Table 5 - 2. Adenoviruses are double - stranded DNA v iru ses (Group I of Baltimore classification) . Its virion size ranges from 70 to 140 nm in diameter, and the isoelectic 169 point ( IEP ) ranges between 3.5 and 4.5 (Xagoraraki et al. 2014). Removal of adenoviruses in full - scale wastew ater treatment plants has be en investigated in previous studies. In conve n tional wastewater treatment plants, the reported removal of adenoviruses ranged from 1.3 logs to 2.4 logs (Haramoto et al. 2007; Hewi tt et al. 2011; Katayama et al. 2008), while in MBR systems, the removal has a higher range from 3 .4 logs to 6.3 logs (Kuo et al. 2010; Simmons et al. 2011; Simmons and Xagoraraki 2011). Most previous studies in this field emphasized the overall virus removal during the wastewater treatment process . W hile the sorption kinetics of viruses in sludge has been demonstrated by on ly a few studies (Clarke et al. 1961; Vilker et al. 1980) and the mechanisms of virus sorption in activated sludge are rarely illustrated . In order to accurately describe the fate of viruses in wastewater treatm ent systems, a better understanding of virus sorption and desorption is needed. Ads orption and desorption isotherms of human adenovirus (HAdV) have been es tablished in soils (Wong et al. 2013), while estigated yet. The objective of this study is to investigate adsorption and des orption of human adenovirus with sludge particles . 5.2. Material and Methods 5. 2. 1. Human adenovirus preparation Human adenovirus 40 was selected for this study, and it was pr opagated in A549 cell lines (ATCC, VR - 846). The d etailed procedure of virus incubation was described in our previous study (Yin et al. 2015). 170 5. 2. 2. Sludge sampling and processing Fresh primary and secondary sludge samples were collected from the wastew ater treatment plant, East Lansing, MI , and kept at 4 °C before use. Since the primary sludge was very condensed, it was diluted by 30 times before processing. Measurement of total suspended solids (TSS) was conducted in duplicate by passing 40 mL well - mix ed sludge sample through 0.45 µm filter, then incubating the filter at 108°C for 1 hour. Dissolved organic carbon (DOC) was measured using TOC analyzer (OI Analytical). The results are present in T able 5 - 3. 5. 2.3. DNA extraction and qPCR assay Extraction of virus DNA was implemented using MagNa Pure Compact System automatic machine and Nucleic Acid Isolation Kits (Roche Applied Sciences). Carrier RNA (Qiagen, Valencia, CA) was used to increase the efficiency of DNA recovery. The DNA was stored at - 80 °C im mediately after extraction. Quantification of virus was conducted in triplicate afterward using qPCR (Roche Light Cycler), of which the assay (sequence of primers and probe) were adopted from Xagoraraki et al. (2007). Crossing point (C p ) values were genera ted by the Light Cycler program. Virus concentrations were determined based on C p values and previously developed standard curves. Inhibition test was conducted by spiking same amount of HAdV to liquid phage of sludge and DI water, and then measuring virus concentration by qPCR. No significant inhibition effect was found. 5.2.4 . Equilibrium time determination A rate study was conducted to determine time needed to reach equilibrium. Sludge sample was well mixed then diluted to the solid/liquid (S/L) ratio of 1:20000 171 as dry weight. 1 mL of virus solution (~ 10 7 ) was added to 9 mL of diluted sludge and mixed by a tumbler at 20 rpm at room temperature. At the end of 12, 24, 48 and 72 h tumbling periods, vials w ere withdrawn and centrifuged at 3500 rpm and the concentration of HAdV in the supernatant was measured by qPCR accordingly. For the desorption experiments, equilibrium - adsorbed solids was prepared in the same way. 5.2.5. Optimal solid/liquid ratio determination Based on MLSS data fr om S ection 5. 2 .2 , s ludg e samples were diluted with DI water pH = 7) to 9 mL in glass tubes with the S/L ratio of 1:4000, 1:20000 and 1:40000 as dry weight. Then 1 mL of virus solutions with HAdV concentration of ~ 10 10 and ~ 10 6 were added to diluted sludge. The tubes were pla ce on a tumbler and rotated at 20 rpm for 72 h. The sludge - virus solution was centrifuged at 3500 rpm and the concentration of HAdV in the supernatant was measured by qPCR accordingly. 5. 2.6. HAdV adsorption The secondary sludge samples were well - mixed and settled for 1 h. The supernatant was filtered through 0.22 µm syringe - driven PVDF filter units, and the pH was then adjusted to 7. S ludg e samples were diluted with DI water (pH = 7) and the sludge filtrate respectively to 9 mL in glass tubes with the S /L ratio of 1: 1 0000 and 1: 2 0000 as dry weight. Then 1 mL of virus solutions with HAdV concentration of ~ 10 10 and ~ 10 6 were added to diluted sludge. The samples were tumbled at 20 rpm until the equilibrium was reached (determined in S ection 5. 2.4), and the n centrifuged at 3500 rpm for 10 min. HAdV concentration in the supernatant was quantified using qPCR. 172 5.2.7. Sorption isotherm experiments Human adenovirus stock was diluted with DI water to series of desired concentration (10 6 - 10 10 virus/ml) in 10 mL tu bes. Based on the MLSS concentration measured S ection 5. 2.2 , proper amount of well - mixed sludge was added to virus suspension, so the S/L ratio of the solution matched the optima l ratio determined in S ection 5.2. 5. Tubes were mixed on a tumbler at 20 rpm f or 48 h. After the equilibrium period was reached, vials were centrifuged at 3500 rpm and then the supernatant was collected to measurement assay. For each concentration of virus, a control tube without sludge was made in order to monitor the loss of virus due to inactivation and sorption to the tubes. Control tubes were treated the way as the experimental tubes. This experiment was implemented in duplicate. The virus concentration on solids was determined according to mass balance: [1] Where , C I , C L , and C S are the virus concentration in liquid phase of control (virus/mL), in the experimental liquid phase (virus/mL), and sorbed to the solid (virus/g), respectively, and M is the total mass of solid per unit volume of virus suspension (g/mL) in each e xperimental tube. Data from sorption experiment were then illustrated by Freundlich equation: [2] Where, K F is the Freundlich constant, which can be used to estimate the adsorption capacity of sorbent; n is the slope of the curve, which is rel ated to the adsorption intensity (Voice and Weber, 1983). 5.2.8. Sequential desorption experiments Desorption experiment was implemented as follow: experimental tubes with ~10 6 173 virus and optimal S/L ratio were made as determin ed in S ection 5. 2. 5. After re aching equilibrium, the supernatant was removed after centrifuge at 3500 rpm, and replaced with DI water. Then the tubes were placed on tumbler and rotated at 20 rpm until equilibrium (determine d in S ection 5. 2.4 ). The procedure was repeated 10 times. The experiment was carried out in duplicate, and control tubes were made accordingly. 5.3. Results and Discussion 5.3. 1. Equilibrium time and optimal S/L ratio Virus concentration in supernatant was 5.09 logs, 4.68 logs, 4.96 logs after 24 h, 48 h and 72 h tumbling. For desorption, virus concentration in the supernatant was 4.29 logs and 4.37 logs after 12 h and 24 h tumbling. As a result, 48 h and 12 h were s elected as equilibrium time for ad sorption and desorption experiment s , respectively. Arraj et al. ( 2005) observed the different sorption behavior of five types of viruses in mixed liquor of activated sludg e, and in most cases of their experiment s , it took 48 hours for virus concentration to reach constant in the aeration tank. Comparatively, it only too k 45 minutes for coxsackie A9 virus concentration reducing by > 99% in activated sludge. Malina et al. (1975) reported that the decrease of poliovirus in activate sludge supernatant became insignificant 1 hour after initial spiking. It has been suggested t hat properties of viruses and sorbents are the key factor governing virus sorption behavior (Gerba et al. 1980; Goyal and Gerba 1979; Chattopadhy ay et al. 2002; Vilker 1981). As shown in Table 5 - 4 , when S/L ratio = 1:4000, virus concentration was 2.72 lo gs and 7.46 logs, both of which were more than 1 logs lower than the control. In contrast, 0.69 log and 0.59 log of HAdV was lost due to adsorption at the ratio 1:20000. Further dilution to the ratio of 1:40000 led to negligible adsorption. In this study, S/L ratio of 1:20000 was applied in all experiments. 174 5.3.2. HAdV adsorption to sludge using DI water and sludge filtrate as solute As shown in Table 5 - 5 , hi gher solid content led to greater virus adsorption. This is because more adsorption sites are availa ble for viruses to attach. Previous studies have demonstrated that o rganic substances, such as proteinaceous matter generally inhibit virus adsorption by competing sorption sites with viruses ( Oza and Chaudhuri 1977; Bales et al. 1993; Lo and Sproul 1977; Stagg et al. 1977). Pieper et al. (1997) observed sewage - derived organic matter decreased adsorption of PRD 1 coliphage to aquifer gains. Bradford et al. ( 2006) found that presence of manure retarded the adsorption of MS - 2 and However, our d ata clearly shows that virus adsorption to sludge particles was stronger in sludge filtrate (DOC = 8.1 mg/L) compared to DI water, as less HAdV was present in the liquid phase of the mixture. The results suggest that the effect of some other components in activated sludge, presumably inorganic ions such as Ca 2+ and Mg 2+ , supersedes the effect of organic matter, resulted in a net enhancement in virus adsorption. Both HAdV and activated sludge particles are negatively charge at neutral pH in aqueous environme nt (Liao et al. 2000; Steiner et al. 1976; Michen and Graule 2010), and it causes a repulsion force between each other. The shielding effect is more profound at higher ionic strength, which prevents virus and sludge particles from interacting with each oth er. The electrostatic repulsion between particles is weakened, and thus adsorption is strengthened. Furthermore, according to DLVO theory the electric double layer around viruses and sorbents is suppressed because of high ionic strength. Virus and sludge p articles have greater chance to get close to each other, and thus adsorption is increased. The composition of activated sludge is complex and could be prominently varied. M u ltivalent ions such as Ca 2+ are much more effective to alter virus sorption compare d to monovalent, such as Na + (Bales et al. 1991; Redm an et al. 1999; Lance and Gerba 1984). The effect of organic matter on virus sorption is dependent on its 175 properties (Zhuang and Jin 2003 b ). More research is needed to understand the contribution of each component in wastewater to virus adsorption. Mathematic models may be built to describe the virus adsorption as a function of ionic strength and the concentration of organic matter. 5.3.3. Adsorption isotherm of HAdV Sorption isotherm curves of mixed l iquor sludge and primary sludge are plotted in Figure 5 - 1. The K F values for the two types of sludge were 3.66×10 4 and 3.92×10 4 , while n values were 1.04 and 1.01, respectively. No significant difference was found between the two types of sludge when t tes t was applied on K F and n values. The results indicate that these two types of sludge particles exhibited similar sorption capacity and intensity despite the fact that the organic concentration in samples with primary sludge (DOC = 2.9 mg/L) was much highe r compared to samples with secondary sludge (DOC = 0.15 mg/L) . The effect of organic matter might be offset by other components in wastewater water, such as ions, as described above. Wong et al. (2013) established isotherms for soils with 2% and 8% organ ic content, in which the K F values were 2.2×10 3 and 5.0×10 2 , while the n values were 1.04 and 1.07, respectively. Comparing to our data, it suggests that sludge in wastewater treatment process may have a higher capacity, but similar intensity to adsorb ade novirus comparing to soils. Clarke et al. (1961) developed an isotherm for poliovirus (type I) in activated sludge with the parameters K F = 7.4×10 2 , and n = 1.02 (isotherm curve was re - plotted based on the readings from the original graph). Vilker et al. ( 1980) also conducted sorption experiments on poliovirus concentration in activated sludge and obtained similar parameters (K F = 7.4×10 2 , n = 1), based on their isotherm curve . It suggests that poliovirus has less affinity to sludge 176 particles comparing to adenovirus as the K F value of poliovirus is lower. Farrah et al. (1978) reported poliovirus showed higher adsorptive capacity than rotavirus in activated sludge, but their sorption experiment only included 5 min mixing of sludge floc and virus for ad sorption. Freundlich isotherms have been widely used in other aspects related to virus sorption. Bitton et al. (1976) used Freundlich isotherm to describe sorption behavior of poliovirus to magnetite in water and wastewater, and they found virus ads orption was affect by cations, but not by variation of pH from 5 to 9. Burge and Enkiri (1978) - 174 with 5 types of soils. Decent accordance to Freundlich isotherm was observed in 4 types of soils, while higher content of organic matter that might block adsorption was attributed to the poor correlation in the other soil. With the assistance of Freundlich isotherm, Moore et al. (1981) reported that poliovirus adsorption to soils and minerals was negatively correlated with organic c ontent and negative surface charge on the substrates. 5.3.4. HAdV desorption from sludge particles Figure 5 - 2 shows the percentage of HAdV desorbed from each sequential desorption experiment. In both primary and secondary sludge, around 10% of HAdV was detached from sludge particles, and then the rate became slower. The cumulative percent over the 4 sequential desorption experiment was 23.8% and 16.9%, respectively. Statistic al analys is showed no significant difference (p > 0.05) of HAdV desorption betwe en the two types of sludge. Virus concentration in the liquid phase was below detention limit after the 5 th sequential experiment. Our data suggests that desorption of HAdV from sludge particles was not very significant, and it is consistent to previous st udies: Clarke et al. (1961) found only a small fraction 177 adsorbed poliovirus detached from sludge particles and then suggested the sludge - virus matrix was stable. Pepper et al. (2006) reported less than 8% of indigenous coliphage was washed out from biosoli ds - soil matrix. Bitton et al. (1984) suggested sludge - soil matrix showed strong capacity to retain enteroviruses. Hurst and Brashear (1987) also reported similar results that no prominent desorption of viruses from sludge after land application. In the fut ure, desorption isotherms of viruses need to be built to further evaluate the reversibility of virus adsorption to sludge particles. 5.4. Implications In this study, we found adsorption of human adenovirus in primary and secondary sludge was well accordan t to Freundlich isotherms. The two types of sludge demonstrated very similar behavior of adsorbing hu man aden ovirus. Overall, virus desorption from sludge particles was insignificant. More HAdV was desorbed from primary sludge than from secondary sludge, b ut the difference was not statistically significant. Greater adsorption of HAdV was observed when liquid phase of activated sludge was used as solute compared to DI water, and it might be a result of compound effect of the inorganic ions (enhance virus ads orption) and organic substances (inhibit virus adsorption). Although removal of viruses by activated sludge has been frequently reported, mechanisms of virus adsorption to sludge particles and the role of sludge components are still unclear. More studies a re needed to further evaluate the fate and transport dynamics of viruses in wastewater systems. Sorption and desorption isotherms in dewater sludge/biosolids should be established since dewatered sludge will be transferred from wastewater to land applicati on. Virus transport to the water 178 environment is likely to be governed by desorption. Complexity and diversity of wastewater properties is the major obstacle to predict virus transport. The impact of each wastewater component on virus adsorption and desorpt ion should be isolated and link to virus surface properties, such as morphology, hydrophobicity, and isoelectric point. 179 APPENDIX 180 Table 5 - 1. Virus partitioning/removal due to sorption in activated sludge Virus Solids type Virus partitioning/ removal due to sorption Reference Coxsaekie virus Activated sludge 99.99% removal after 6 h Clarke et al. 1961 Poliovirus Echovirus Coxsackievirus Activated sludge 67% - 99.8% on solids depending on virus type Gerba et al. 1980 Poliovirus Activated sl udge 21% - 45% on solids depending on solid concentration Vilker and Kamdar 1980 T4, f2 Activated sludge 0.8% - 22% removal Zheng and Liu 2007 Hepatitis A virus, poliovirus, rotavirus, MS2, X174 Activated sludge 0% - 99.6% on solids depending on virus ty pe Arraj et al. 2005 Poliovirus Activated sludge ~ 85% on solids Balluz et al. 1977 f2 coliphage Activated sludge Distribution of virus in solid and liquid phase: 18:82 Balluz et al. 1978 Poliovirus, rotavirus Activated sludge 68.4% - 98.4% adsorbed on solids depending on virus type Farrah et al. 1978 Poliovirus Activated sludge > 99% on solids Malina et al. 1975 Rotavirus Raw sewage 55% on solids Rao et al. 1987 Primary sludge 42% on solids Secondary sludge (aeration chambers) 92% on solids F inal effluent 88% on solids Enterovirus Secondary sludge 83% - 99% adsorbed on solids Moore et al. 1978 Human adenovirus, human enterovirus, norovirus Secondary sludge (membrane tank) Virus concentration in the settled sludge is 3 4 logs higher compar ed to the filtered sludge supernatant Simmons et al. 2011 181 Table 5 - 2. Virus desorption from sludge/biosolids Virus Solids type Virus desorption Reference Poliovirus Activated sludge A small fraction of virus desorbed from sludge Clarke et al. (1961) Poliovirus Sludge after land application No significant desorption Hurst and Brashear (1987) Coliphage Biosolids soil matrix Less than 8% Pepper et al. (2006) Enteroviruses Sludge - soil matrix No significant desorption Bitton et al. (1984) 182 Ta ble 5 - 3. TSS and DOC in primary and secondary sludge TSS, g/L DOC, mg/L Primary sludge (diluted by 30 times) 0.71 40.6 Secondary sludge 2.66 8.10 183 Table 5 - 4 . Virus concentration in supernatant with different S/L ratio Original virus conc. ~ 5 log s ~ 9 logs Control 4.31 logs 8.77 logs S/L = 1:4000 2.72 logs 7.46 logs S/L = 1:20000 3.62 logs 8.18 logs S/L = 1:40000 4.38 logs 8.65 logs 184 Table 5 - 5 . Comparison of DI water and sludge filtrate as solute for HAdV adsorption# Original virus conc. ~ 9 logs ~ 5 logs Solute DI water Sludge filtrate DI water Sludge filtrate S/L = 1:10000 7.21 logs 6.40 logs 2.93 logs BDL* S/L = 1:20000 8.65 logs 8.27 logs 4.40 logs 3.31 logs #Virus concentration in the table represents the log virus concentration in the liquid phase *BDL: below detection limit 185 Figure 5 - 1 . Ad orption isotherm curves (1) prima ry sludge; (2) secondary sludge* * Filled and hollow cycles are replicates 186 Figure 5 - 2 . Percentage of HAdV desorbed from sludge particles in sequential experiments: (1) Primary sludge; (2) Secondary sludge 187 REFERENCES 188 REFERENCES Abzug, M. J., et al. (2003). Double blind placebo - controlled trial of pleconaril in infants with enterovirus meningitis. The Pediatric Infectious Dise ase Journal , 22 (4), 335 - 340. Arraj, A., Bohatier, J., Laveran, H., & Traore, O. (2005). Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants. Journal of Applied Microbiology , 98 (2), 516 - 524. Aslan, A., Xagoraraki, I. , Simmons, F. J., Rose, J. B., & Dorevitch, S. (2011). Occurrence of adenovirus and other enteric viruses in limited contact freshwater recreational areas and bathing waters. Journal of Applied Microbiology , 111 (5), 1250 - 1261. Bales, R. C., Hinkle, S. R., Kroeger, T. W., Stocking, K., & Gerba, C. P. (1991). Bacteriophage adsorption during transport through porous media: Chemical perturbations and reversibility. Environmental Science and Technology , 25 (12), 2088 - 2095. Bales, R. C., Li, S., Maguire, K. M., Ya hya, M. T., & Gerba, C. P. (1993). MS - 2 and poliovirus transport in porous media: Hydrophobic effects and chemical perturbations. Water Resource Research , 29 (4), 957 - 963. Bales, R. C., Li, S., Maguire, K. M., Yahya, M. T., Gerba, C. P., & Harvey, R. W. (19 95). Virus and bacteria transport in a sandy aquifer, Cape Cod, MA. Groundwater , 33 (4), 653 - 661. Ballester, N. A., & Malley, J. P. (2004). Sequential disinfection of adenovirus type 2 with UV - chlorine - chloramine. Journal (American Water Works Association) , 97 - 103. Balluz, S. A., Jones, H. H., & Butler, M. (1977). The persistence of poliovirus in activated sludge treatment. Journal of hygiene , 78 (02), 165 - 173. Balluz, S. A., Butler, M., & Jones, H. H. (1978). The behaviour of f2 coliphage in activated sludge treatment. Journal of hygiene , 80 (02), 237 - 242. Bitton, G., & Harvey, R. W. (1992). Transport of pathogens through soils and aquifers. Environmental Microbiology , 103 - 124. Bitton, G., Pancorbo, O. C., & Farrah, S. R. (1984). Virus transport and survival a fter land application of sewage sludge. Applied Environmental Microbiology , 47 (5), 905 - 909. Bitton, G. (1975). Adsorption of viruses onto surfaces in soil and water. Water Research , 9 (5), 473 - 484. Bitton, G., Pancorbo, O., & Gifford, G. E. (1976). Factors affecting the adsorption of polio virus to magnetite in water and wastewater. Water Research , 10 (11), 973 - 980. 189 Bixby, R. L., & O'Brien, D. J. (1979). Influence of fulvic acid on bacteriophage adsorption and complexation in soil. Applied Environmental Micro biology , 38 (5), 840 - 845. Blanc, R., & Nasser, A. (1996). Effect of effluent quality and temperature on the persistence of viruse in soil. Water Science and Technology , 33 (10), 237 - 242. Boche, R. D., & Quilligan, J. J. (1966). Adsorption to Glass and Specif ic Antibody Inhibition of Iodine125 Labeled Influenza Virus. The Journal of Immunology , 97 (6), 942 - 950. Bofill - Mas, S., Albinana - Gimenez, N., Clemente - Casares, P., Hundesa, A., Rodriguez - Manzano, J., Allard, A., Calvo, M., & Girones, R. (2006). Quantificat ion and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices. Applied Environmental Microbiology , 72 (12), 7894 - 7896. Bradford, S. A., Tadassa, Y. F., & Jin, Y. (2006). Transport of coliphage in the presence and absence of manure su spension. Journal of Environmental Quality , 35 (5), 1692 - 1701. Bradley, I., Straub, A., Maraccini, P., Markazi, S., & Nguyen, T. H. (2011). Iron oxide amended biosand filters for virus removal. Water Research , 45 (15), 4501 - 4510. Burge, W. D., & Enkiri, N. K . (1978). Virus adsorption by five soils. Journal of Environmental Quality , 7 (1), 73 - 76. Cao, H., Tsai, F. T. C., & Rusch, K. A. (2009). Impact of Salinity on MS - 2 Sorption in Saturated Sand Columns Fate and Transport Modeling. Journal of Environmental Eng ineering , 135 (10), 1041 - 1050. Carlson Jr, G. F., Woodard, F. E., Wentworth, D. F., & Sproul, O. J. (1968). Virus inactivation on clay particles in natural waters. Journal (Water Pollution Control Federation) , R89 - R106. Castignolles, N., Petit, F., Mendel, I., Simon, L., Cattolico, L., & Buffet - Janvresse, C. (1998). Detection of adenovirus in the waters of the Seine River estuary by nested - PCR. Molecular and Cellular Probes , 12 (3), 175 - 180. Chang, J. C., Ossoff, S. F., Lobe, D. C., Dorfman, M. H., Duma is, C. M., Qualls, R. G., & Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. Applied Environmental Microbiology , 49 (6), 1361 - 1365. Chattopadhyay, S., & Puls, R. W. (1999). Adsorption of bacteriophages on clay minerals. Env ironmental Science and Technology , 33 (20), 3609 - 3614. Chattopadhyay, D., Chattopadhyay, S., Lyon, W. G., & Wilson, J. T. (2002). Effect of surfactants on the survival and sorption of viruses. Environmental Science and Technology , 36 (19), 4017 - 4024. Chaudhu ri, M., Koya, K. V. A., & Sriramulu, N. (1977). Some notes on virus retention by sand. The Journal of General and Applied Microbiology , 23 (6), 337 - 344. Chu, Y., Jin, Y., & Yates, M. V. (2000). Virus transport through saturated sand 190 columns as affected by d ifferent buffer solutions. Journal of Environmental Quality , 29 (4), 1103 - 1110. Chu, Y., Jin, Y., Flury, M., & Yates, M. V. (2001). Mechanisms of virus removal during transport in unsaturated porous media. Water Resource Research , 37 (2), 253 - 263. Chu, Y., J in, Y., Baumann, T., & Yates, M. V. (2003). Effect of soil properties on saturated and unsaturated virus transport through columns. Journal of Environmental Quality , 32 (6), 2017 - 2025. Clark, K. J., Sarr, A. B., Grant, P. G., Phillips, T. D., & Woode, G. N. (1998). In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus. Veterinary Microbiology , 63 (2), 137 - 146. Clarke, N. A., Stevenson, R. E., Chang, S. L., & Kabler, P. W. (1961). Removal of enteric v iruses from sewage by activated sludge treatment. American Journal of Public Health and the Nations Health , 51 (8), 1118 - 1129. Cliver, D. O. (1968). Virus interactions with membrane filters. Biotechnology and Bioengineering , 10 (6), 877 - 889. Cookson Jr, J. T . (1969). Mechanism of virus adsorption on activated carbon. Journal (American Water Works Association) , 52 - 56. Da Silva, A. K., Le Saux, J. C., Parnaudeau, S., Pommepuy, M., Elimelech, M., & Le Guyader, F. S. (2007). Evaluation of removal of noroviruses du ring wastewater treatment, using real - time reverse transcription - PCR: different behaviors of genogroups I and II. Applied Environmental Microbiology , 73 (24), 7891 - 7897. Dowd, S. E., Pillai, S. D., Wang, S., & Corapcioglu, M. Y. (1998). Delineating the spec ific influence of virus isoelectric point and size on virus adsorption and transport through sandy soils. Applied Environmental Microbiology , 64 (2), 405 - 410. Drewry, W. A., & Eliassen, R. (1968). Virus movement in groundwater. Journal (Water Pollution Cont rol Federation) , R257 - R271. Eischeid, A. C., Meyer, J. N., & Linden, K. G. (2009). UV disinfection of adenoviruses: molecular indications of DNA damage efficiency. Applied Environmental Microbiology , 75 (1), 23 - 28. Elliott, L. F., & Ellis, J. R. (1977). Bac terial and viral pathogens associated with land application of organic wastes. Journal of Environmental Quality , 6 (3), 245 - 251. Englande Jr, A. J. (1983). Incidence and fate of viruses in sludges. In Sludge Characteristics and Behavior , 280 - 293. Springer N etherlands. Farkas, T., Sestak, K., Wei, C., & Jiang, X. (2008). Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae. Journal of Virology , 82 (11), 5408 - 5416. Farrah, S. R., Goyal, S. M., Gerba, C. P., Conklin, R. H., & Smith, E. M. (1978). 191 Comparison between adsorption of poliovirus and rotavirus by aluminum hydroxide and activated sludge flocs. Applied Environmental Microbiology , 35 (2), 360 - 363. Farrah, S. R., Shah, D. O., & Ingram, L. O. (1981). Effects of chaotropic and antichaotropic agents on elution of poliovirus adsorbed on membrane filters. Proceedings of the National Academy of Sciences , 78 (2), 1229 - 1232. Gerba, C. P., & Schaiberger, G. E. (1975). Effect of particulates on virus survival in seawater. Journal (Wa ter Pollution Control Federation) , 93 - 103. Gerba, C., Wallis, C., & Melnick, J. (1975). Viruses in water: the problem, some solutions. Environmental Science and Technology , 9 (13), 1122 - 1126. Gerba, C. P. (1984). Applied and theoretical aspects of virus ads orption to surfaces. Advances in Applied Microbiology , 30 , 133 - 168. Gerba, C. P., Goyal, S. M., Hurst, C. J., & Labelle, R. L. (1980). Type and strain dependence of enterovirus adsorption to activated sludge, soils and estuarine sediments. Water Research , 14 (9), 1197 - 1198. Gerba, C. P., Goyal, S. M., Cech, I., & Bogdan, G. F. (1981). Quantitative assessment of the adsorptive behavior of viruses to soils. Environmental Science and Technology , 15 (8), 940 - 944. Gerba, C. P. (2007). Virus occurrence and survival in the environmental waters. A Bosch, Human Viruses in Water, 17 , 91 - 108. Goyal, S. M., & Gerba, C. P. (1979). Comparative adsorption of human enteroviruses, simian rotavirus, and selected bacteriophages to soils. Applied Environmental Microbiology , 38 (2), 241 - 247. Grant, S. B., List, E. J., & Lidstrom, M. E. (1993). Kinetic analysis of virus adsorption and inactivation in batch experiments. Water Resource Research , 29 (7), 2067 - 2085. Guttman - Bass, N., & Catalano - Sherman, J. (1986). Humic acid interference w ith virus recovery by electropositive microporous filters. Applied Environmental Microbiology , 52 (3), 556 - 561. Han, J., Jin, Y., & Willson, C. S. (2006). Virus retention and transport in chemically heterogeneous porous media under saturated and unsaturated flow conditions. Environmental Science and Technology , 40 (5), 1547 - 1555. Haramoto, E., Katayama, H., Oguma, K., & Ohgaki, S. (2007). Quantitative analysis of human enteric adenoviruses in aquatic environments. Journal of Applied Microbiology , 103 (6), 2153 - 2159. Hewitt, J., Leonard, M., Greening, G. E., & Lewis, G. D. (2011). Influence of wastewater treatment process and the population size on human virus profiles in wastewater. Water Research , 45 (18), 6267 - 6276. Hurst, C. J., Gerba, C. P., Lance, J. C., & Rice, R. C. (1980). Survival of enteroviruses 192 in rapid - infiltration basins during the land application of wastewater. Applied Environmental Microbiology , 40 (2), 192 - 200. Hurst, C. J., & Brashear, D. A. (1987). Use of a vacuum filtration technique to study leaching of indigenous viruses from raw wastewater sludge. Water Research , 21 (7), 809 - 812. Ivanova, V. T., Katrukha, G. S., Timofeeva, A. V., Ilyna, M. V., Kurochkina, Y. E., Baratova, L. A., ... & Ivanov, V. F. (2011, April). The sorption of influenza vir uses and antibiotics on carbon nanotubes and polyaniline nanocomposites. In Journal of Physics: Conference Series (Vol. 291, No. 1, p. 012004). IOP Publishing. Jewett, D. G., Logan, B. E., Arnold, R. G., & Bales, R. C. (1999). Transport of Pseudomonas fluor escens strain P17 through quartz sand columns as a function of water content. Journal of Contaminant Hydrology , 36 (1), 73 - 89. Jin, Y., Pratt, E., & Yates, M. V. (2000). Effect of mineral colloids on virus transport through saturated sand columns. Journal o f Environmental Quality , 29 (2), 532 - 539. Jørgensen, P. H., & Lund, E. (1986). Transport of Viruses from Sludge Application Sites. In Processing and Use of Organic Sludge and Liquid Agricultural Wastes , 215 - 224. Springer Netherlands. Katayama, H., Haramoto, E., Oguma, K., Yamashita, H., Tajima, A., Nakajima, H., & Ohgaki, S. (2008). One - year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan. Water Research , 42 (6), 1441 - 1448. Kim, T. D ., & Unno, H. (1996). The roles of microbes in the removal and inactivation of viruses in a biological wastewater treatment system. Water Science and Technology , 33 (10), 243 - 250. Ko, G., Cromeans, T. L., & Sobsey, M. D. (2005). UV inactivation of adenoviru s type 41 measured by cell culture mRNA RT - PCR. Water Research , 39 (15), 3643 - 3649. Kuo, D. H. W., Simmons, F. J., Blair, S., Hart, E., Rose, J. B., & Xagoraraki, I. (2010). Assessment of human adenovirus removal in a full - scale membrane bioreactor treating municipal wastewater. Water Research , 44 (5), 1520 - 1530. Lance, J. C., Gerba, C. P., & Melnick, J. L. (1976). Virus movement in soil columns flooded with secondary sewage effluent. Applied Environmental Microbiology , 32 (4), 520 - 526. Lance, J. C., & Gerba, C. P. (1984). Effect of ionic composition of suspending solution on virus adsorption by a soil column. Applied Environmental Microbiology , 47 (3), 484 - 488. Landry, E. F., Vaughn, J. M., & Penello, W. F. (1980). Poliovirus retention in 75 - cm soil cores after sewage and rainwater application. Applied Environmental 193 Microbiology , 40 (6), 1032 - 1038. Liao, B. Q., Allen, D. G., Droppo, I. G., Leppard, G. G., & Liss, S. N. (2001). Surface properties of sludge and their role in bioflocculation and settleability. Water Research , 35 (2), 339 - 350. Lipson, S. M., & Stotzky, G. (1985). Specificity of virus adsorption to clay minerals. Canadian Journal of Microbiology , 31 (1), 50 - 53. Lo, S. H., & Sproul, O. J. (1977). Polio - virus adsorption from water onto silicate minerals. W ater Research , 11 (8), 653 - 658. Madaeni, S. S., Fane, A. G., & Grohmann, G. S. (1995). Virus removal from water and wastewater using membranes. Journal of Membrane Science , 102 , 65 - 75. Malina Jr, J. F., Ranganathan, K. R., Sagik, B. P., & Moore, B. E. (1975 ). Poliovirus inactivation by activated sludge. Journal (Water Pollution Control Federation) , 2178 - 2183. Marshall, G. S. (2009). Rotavirus disease and prevention through vaccination. The Pediatric Infectious Disease Journal , 28 (4), 351 - 364. Martone, W. J., Hierholzer, , J. C., Keenlyside, R. A., Fraser, D. W., D'Angelo, L. J., & Winkler, W. G. (1980). An outbreak of adenovirus type 3 disease at a private recreation center swimming pool. American Journal of Epidemiology , 111 (2), 229 - 237. Michen, B., & Graule , T. (2010). Isoelectric points of viruses. Journal of Applied Microbiology , 109 (2), 388 - 397. Moore, B. E., Sagik, B. P., & Sorber, C. A. (1978). Land application of sludges: minimizing the impact of viruses on water resources. Risk Assessment and Health E ffects of Land Application of Municipal Wastewater and Sludges, BP Sagik, and CA Sorber, Eds., University of Texas, San Antonio, TX , 154 - 167. Moore, R. S., Taylor, D. H., Sturman, L. S., Reddy, M. M., & Fuhs, G. W. (1981). Poliovirus adsorption by 34 miner als and soils. Applied Environmental Microbiology , 42 (6), 963 - 975. Monpoeho, S., Maul, A., Mignotte - Cadiergues, B., Schwartzbrod, L., Billaudel, S., & Ferre, V. (2001). Best viral elution method available for quantification of enteroviruses in sludge by bo th cell culture and reverse transcription - PCR. Applied Environmental Microbiology , 67 (6), 2484 - 2488. Monpoeho, S., Maul, A., Bonnin, C., Patria, L., Ranarijaona, S., Billaudel, S., & Ferre, V. (2004). Clearance of human - pathogenic viruses from sludge: stud y of four stabilization processes by real - time reverse transcription - PCR and cell culture. Applied Environmental Microbiology , 70 (9), 5434 - 5440. Moore, R. S., Taylor, D. H., Sturman, L. S., Reddy, M. M., & Fuhs, G. W. (1981). Poliovirus adsorption by 34 mi nerals and soils. Applied Environmental Microbiology , 42 (6), 963 - 975. 194 National Research Council (NRC). (2002). Biosolids applied to land, National Academies, Washington, DC. Oza, P. P., & Chaudhuri, M. (1975). Removal of viruses from water by sorption on c oal. Water Research , 9 (8), 707 - 712. Oza, P. P., & Chaudhuri, M. (1976). Virus - coal sorption interaction. J. Environ. Eng. Div., Am. Soc. Civ. Eng., (United States) , 102 . Penrod, S. L., Olson, T. M., & Grant, S. B. (1996). Deposition kinetics of two viruses in packed beds of quartz granular media. Langmuir , 12 (23), 5576 - 5587. Pepper, I. L., Brooks, J. P., & Gerba, C. P. (2006). Pathogens in biosolids. Advances in Agronomy , 90 , 1 - 41. Pham, M., Mintz, E. A., & Nguyen, T. H. (2009). Deposition kinetics of bacter iophage MS2 to natural organic matter: Role of divalent cations. Journal of colloid and interface science , 338 (1), 1 - 9. Pieper, A. P., Ryan, J. N., Harvey, R. W., Amy, G. L., Illangasekare, T. H., & Metge, D. W. (1997). Transport and recovery of bacterioph age PRD1 in a sand and gravel aquifer: Effect of sewage - derived organic matter. Environmental Science and Technology , 31 (4), 1163 - 1170. Poletika, N. N., Jury, W. A., & Yates, M. V. (1995). Transport of Bromide, Simazine, and MS 2 Coliphage in a Lysimeter Containing Undisturbed, Unsaturated Soil. Water Resource Research , 31 (4), 801 - 810. Powelson, D. K., Simpson, J. R., & Gerba, C. P. (1990). Virus transport and survival in saturated and unsaturated flow through soil columns. Jour nal of Environmental Quality , 19 (3), 396 - 401. Powelson, D. K., Simpson, J. R., & Gerba, C. P. (1991). Effects of organic matter on virus transport in unsaturated flow. Applied Environmental Microbiology , 57 (8), 2192 - 2196. Powelson, D. K., & Mills, A. L. (1 996). Bacterial enrichment at the gas - water interface of a laboratory apparatus. Applied Environmental Microbiology , 62 (7), 2593 - 2597. Preston, D. R., & Farrah, S. R. (1988). Activation thermodynamics of virus adsorption to solids. Applied Environmental Mi crobiology , 54 (11), 2650 - 2654. Puig, M., Jofre, J., Lucena, F., Allard, A., Wadell, G., & Girones, R. (1994). Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Applied Environmental Microbiology , 60 (8), 2963 - 2970. Rao, V. C., Metcalf, T. G., & Melnick, J. L. (1987). Removal of indigenous rotaviruses during primary settling and activated - sludge treatment of raw sewage. Water Research , 21 (2), 171 - 177. Redman, J. A., Grant, S. B., Olson, T. M., Adkins, J. M., Jackson, J. L., Castillo, M. S., 195 & Yanko, W. A. (1999). Physicochemical mechanisms responsible for the filtration and mobilization of a filamentous bacteriophage in quartz sand. Water Research , 33 (1), 43 - 52. Rotbart, H. A. (2000). Viral meningitis. In Seminars in n eurology, 20(3), 277 - 292. Ryan, J. N., Elimelech, M., Ard, R. A., Harvey, R. W., & Johnson, P. R. (1999). Bacteriophage PRD1 and silica colloid transport and recovery in an iron oxide - coated sand aquifer. Environmental Science and Technology , 33 (1), 63 - 73. Sano, D., Matsuo, T., & Omura, T. (2004). Virus - binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide. Applied Environmental Microbiology , 70 (6), 3434 - 3442. Scheuerman , P. R., Gifford, G. E., Overman, A. R., & Bitton, G. (1979). Transport of viruses through organic soils and sediments. Journal of the Environmental Engineering Division , 105 (4), 629 - 640. Schijven, J. F., & Hassanizadeh, S. M. (2000). Removal of viruses by soil passage: Overview of modeling, processes, and parameters. Critical reviews in Environmental Science and Technology , 30 (1), 49 - 127. Schulze - Makuch, D., Guan, H., & Pillai, S. D. (2003). Effects of pH and geological medium on bacteriophage MS2 transpor t in a model aquifer. Geomicrobiology Journal , 20 (1), 73 - 84. Shizas, I., & Bagley, D. M. (2004). Experimental determination of energy content of unknown organics in municipal wastewater streams. Journal of Energy Engineering , 130 (2), 45 - 53. Simmons, F. J., Kuo, D. H. W., & Xagoraraki, I. (2011). Removal of human enteric viruses by a full - scale membrane bioreactor during municipal wastewater processing. Water Research , 45 (9), 2739 - 2750. Simmons, F. J., & Xagoraraki, I. (2011). Release of infectious human ent eric viruses by full - scale wastewater utilities. Water Research , 45 (12), 3590 - 3598. Sobsey, M. D., & Shields, P. A. (1987). Survival and transport of viruses in soils: model studies. Human Viruses in Sediments, Sludges and Soils, 155 - 177. Stagg, C. H., Wal lis, C. R. A. I. G., & Ward, C. H. (1977). Inactivation of clay - associated bacteriophage MS - 2 by chlorine. Applied Environmental Microbiology , 33 (2), 385 - 391. Steiner, A. E., McLaren, D. A., & Forster, C. F. (1976). The nature of activated sludge flocs. Wa ter Research , 10 (1), 25 - 30. Thompson, S. S., & Yates, M. V. (1999). Bacteriophage inactivation at the air - water - solid interface in dynamic batch systems. Applied Environmental Microbiology , 65 (3), 1186 - 1190. Viau, E. J., Lee, D., & Boehm, A. B. (2011). Swi mmer risk of gastrointestinal illness 196 from exposure to tropical coastal waters impacted by terrestrial dry - weather runoff. Environmental Science and Technology , 45 (17), 7158 - 7165. Viau, E., & Peccia, J. (2009). Survey of wastewater indicators and human pat hogen genomes in biosolids produced by class A and class B stabilization treatments. Applied Environmental Microbiology , 75 (1), 164 - 174. Vilker, V. L., Kamdar, R. S., & Frommhagen, L. H. (1980). Capacity of activated sludge solids for virus adsorption. Che mical Engineering Communications , 4 (4 - 5), 569 - 575. Vilker, V. L. (1981). Virus Transport Through Percolating Beds. University of California Water Resources Center . Voice, T. C., & Weber, W. J. (1983). Sorption of hydrophobic compounds by sediments, soils a nd suspended solids I. Theory and background. Water Research , 17 (10), 1433 - 1441. Wallis, C., & Melnick, J. L. (1967). Concentration of enteroviruses on membrane filters. Journal of Virology , 1 (3), 472 - 477. Wan, J., & Wilson, J. L. (1994). Visualization of the role of the gas - water interface on the fate and transport of colloids in porous media. Water Resource Research , 30 (1), 11 - 24. Wan, J., & Tokunaga, T. K. (1997). Film straining of colloids in unsaturated porous media: Conceptual model and experimental t esting. Environmental Science and Technology , 31 (8), 2413 - 2420. Wong, K., Onan, B. M., & Xagoraraki, I. (2010). Quantification of enteric viruses, pathogen indicators, and Salmonella bacteria in class B anaerobically digested biosolids by culture and molec ular methods. Applied Environmental Microbiology , 76 (19), 6441 - 6448. Wong, M., Kumar, L., Jenkins, T. M., Xagoraraki, I., Phanikumar, M. S., & Rose, J. B. (2009). Evaluation of public health risks at recreational beaches in Lake Michigan via detection of e nteric viruses and a human - specific bacteriological marker. Water Research , 43 (4), 1137 - 1149. Wong, K., Voice, T. C., & Xagoraraki, I. (2013). Effect of organic carbon on sorption of human adenovirus to soil particles and laboratory containers. Water Resea rch , 47 (10), 3339 - 3346. Wyn - Gantzer, C., Gawler, A., Girones, R., Höller, C., De Roda Husman, A. M., Kozyra, I., López - Pila., J., Muscillo, M., Nascimento, M., Papageorgiou, G., Rutjes, S., Sellwood, J., Kay, D., & Wyer, M. (2011). Surveillance of adenoviruses and noroviruses in European recreational waters. Water Research , 45 (3), 1025 - 1038. Xagoraraki, I., Yin, Z., & Svambayev, Z. (2014). Fate of viruses in water systems. Jou rnal of Environmental Engineering , 140 (7). 197 Xagoraraki, I., Kuo, D. H. W., Wong, K., Wong, M., & Rose, J. B. (2007). Occurrence of human adenoviruses at two recreational beaches of the great lakes. Applied Environmental Microbiology , 73 (24), 7874 - 7881. Yate s, M. V., Gerba, C. P., & Kelley, L. M. (1985). Virus persistence in groundwater. Applied Environmental Microbiology , 49 (4), 778 - 781. Yeager, J. G., & O'Brien, R. T. (1979). Enterovirus inactivation in soil. Applied Environmental Microbiology , 38 (4), 694 - 7 01. Yin, Z., Tarabara, V. V., & Xagoraraki, I. (2015). Human adenovirus removal by hollow fiber membranes: Effect of membrane fouling by suspended and dissolved matter. Journal of Membrane Science , 482 , 120 - 127. You, Y., Vance, G. F., Sparks, D. L., Zhuang , J., & Jin, Y. (2003). Sorption of MS2 bacteriophage to layered double hydroxides. Journal of Environmental Quality , 32 (6), 2046 - 2053. Zhao, B., Zhang, H., Zhang, J., & Jin, Y. (2008). Virus adsorption and inactivation in soil as influenced by autochthono us microorganisms and water content. Soil Biology and Biochemistry , 40 (3), 649 - 659. Zheng, X., & Liu, J. (2007). Virus rejection with two model human enteric viruses in membrane bioreactor system. Science in China Series B: Chemistry , 50 (3), 397 - 404. Zhuan g, J., & Jin, Y. (2003a). Virus retention and transport through Al - oxide coated sand columns: effects of ionic strength and composition. Journal of Contaminant Hydrology , 60 (3), 193 - 209. Zhuang, J., & Jin, Y. (2003b). Virus retention and transport as influ enced by different forms of soil organic matter. Journal of Environmental Quality , 32 (3), 816 - 823.