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Fixed-film anaerobic digestion

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Fixed-film anaerobic digestion mechanisms of pathogen reduction and impacts on virus adsorption to soil
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Davis, Johnny A
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Adsorption ( jstor )
Anaerobic digestion ( jstor )
Bacteria ( jstor )
Groundwater ( jstor )
Manure ( jstor )
Microorganisms ( jstor )
Pathogens ( jstor )
Soils ( jstor )
Viruses ( jstor )
Wastewater ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF ( lcsh )
Microbiology and Cell Science thesis, Ph. D ( lcsh )
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theses ( marcgt )
non-fiction ( marcgt )

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Thesis (Ph. D.)--University of Florida, 2006.
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Includes bibliographical references.
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Printout.
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Vita.
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by Johnny A. Davis.

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FIXED-FILM ANAEROBIC DIGESTION: MECHANISMS OF PATHOGEN
REDUCTION AND IMPACTS ON VIRUS ADSORPTION TO SOIL















By

JOHNNY A. DAVIS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Johnny A. Davis
































To Ethan and Joshua.















ACKNOWLEDGMENTS

I would like to thank Dr. Samuel R. Farrah, chair of the supervisory committee, for

providing support, research facilities, and guidance towards completion of this body of

work. I would like to extend a sincere appreciation to the co-chair of the supervisory

committee, Dr. Ann C. Wilkie. Her constant persistence and guidance pushed the work

forward to completion. Through our numerous scientific discussions and constant mental

challenging, I have gained a tremendous appreciation for my field of study and the

pursuit of knowledge in general. I have no doubt that our interactions will help me

develop a successful career as an independent scientist. I thank the remaining members

of the supervisory committee, Dr. Ben Koopman, Dr. Peter Kima, and especially, Dr.

Madeline Rasche, for providing valuable insight to career development. I thank my wife,

Nicole Davis, for her patience and support during this long journey. I would like to thank

my parents, Harvey Moffitt and Emma T. Davis, for their wisdom and encouragement. I

would also like to thank Dr. Anne Donnelly for being a mentor and friend. I would also

like to acknowledge David Armstrong and the Dairy Research Unit staff for their help

during various stages of the project. I would like to thank the Department of

Microbiology and Cell Science, faculty and staff. I also extend an appreciation for the

McKnight Doctoral and Southeast Alliance for Graduate Education and the Professoriate

Programs for providing funding during my graduate career.

Lastly, I would like to acknowledge my reunion with my biological parents,

Namon and Debbie Martin, and the introduction to the family I never had the opportunity









to know. During the last four years I have enjoyed learning about my family and I look

forward to our future together as we continue to close the 24-year gap.















TABLE OF CONTENTS

pACKN OW LEDGM ENT S .................................................................................................


ACKNOW LEDGM ENTS............................................................................................ iv

LIST OF TABLES.......................................................................................................... ix

LIST OF FIGURES ........................................................................................................ xi

ABSTRACT...................................................................................................................... xii

CHAPTER

1 INTRODUCTION .....................................................................................................1...

2 PATHOGENIC ORGANISMS IN BOVINE MANURE AND THEIR
REDUCTION BY ANAEROBIC DIGESTION..........................................................4

Pathogenic Organism s in Dairy Cattle M anure .........................................................4...
Zoonotic Bacteria ...............................................................................................5...
M astitic Bacteria............................................................................................... 10
Pathogenic Viruses ........................................................................................... 12
Parasitic Protozoa ............................................................................................. 14
Anaerobic Digestion and Pathogenic Organism s .................................................... 15
Anaerobic Digestion......................................................................................... 15
Anaerobic Digesters ......................................................................................... 16
M icrobial Diversity in Anaerobic Digesters..................................................... 18
Pathogen Decimation during Anaerobic Digestion of Animal Manure ..............20
Sum m ary.....................................................................................................................27
Purpose of Study.................... .....................................................................................28

3 INDICATOR AND PATHOGENIC BACTERIA REDUCTION BY
ANAEROBIC DIGESTION: THE ROLE OF MICROBIAL COMPETITION
AND SUBSTRATE LIM ITATIONS ...................................................................... 29

Introduction.................................................................................................. ...............29
Purpose ............... ................................................................................ .................. 31
M materials and M ethods ...... ........................................... .................. .................... ..32
Dairy Research Unit ............................................... ....................... ............ 32
M anure Handling ............................................... ..... .......................................32









Fixed-film Anaerobic Digester......................................................................... 32
Sample Collection, Characterization, and Preparation..................................... 33
Bacterial Cultures................................................................. ............................ 34
Bacterial Quantification.................................................................................... 34
Anaerobic Conditions....................................................................................... 35
Survival in Whole and Soluble Wastewater Fractions.....................................35
Inhibition and Wastewater Supplementation Studies.......................................36
Statistical Analysis ...........................................................................................37
Results......................................................................................................................... 37
W astewater Characteristics .............................................................................. 37
Growth of Bacteria in Whole and Soluble Fractions of Wastewater ...............38
Total aerobic, anaerobic, and facultative bacteria..................................... 38
Escherichia coli and fecal coliform s......................................................... 39
Enterococcus spp....................................................................................... 40
Salmonella spp. .......................................................................................... 41
Escherichia coli 0 157:H7......................................................................... 41
Staphylococcus aureus.............................................................................. 41
Discussion and Conclusions.................................................................................... 43

4 INDICATOR AND PATHOGENIC BACTERIA REDUCTION DURING
ANAEROBIC DIGESTION: ATTACHMENT TO THE FIXED-FILM ..............56

Introduction............................................................................................................ 56
Purpose .....................................................................................................................56
M materials and M ethods .............. ................................................................ ......... 57
Pilot-Scale Fixed-Film Reactors..........................................................................57
Survival Comparability with Indigenous Fecal Coliforms..................................57
Biofilm Attachm ent Studies ...................................................................................58
Biofilm Sampling and Exam inning ........................................ ......................... 58
Results.............................. ..................................................................................... 59
Fecal Coliform and GFP E. coli Survival................ ......................................59
Biofilm Attachm ent Studies ................................................................................59
Discussion and Conclusions.......................................................................................60

5 BACTERIOPHAGE REDUCTION DURING ANAEROBIC DIGESTION: THE
ROLE OF INDIGENOUS M ICROFLORA ....................................... .......................66

Introduction................................................................................................................. 66
Purpose ...................................................................................................................... 67
M materials and M ethods ............................................ ................................... ................67
Sample Collection, Characterization, and Preparation................................... 67
Bacteriophages ...................................................... ........................................68
Experim ental Design .................. ............................................. ...................... 68
Bacteriophage Quantification.............................................................................. 68
Anaerobic Conditions.................................................................................... 68
Statistical Analysis ................................................... ........................................... 68
Results.........................................................................................................................69









W astewater Characteristics .................................................................................69
Influence of Whole and Soluble Fractions of Wastewater...............................69
Discussion and Conclusions........................................................................... ......69

6 TRANSPORT OF VIRUSES IN SOIL AMENDED WITH ANAEROBICALLY
DIGESTED FLUHSHED DAIRY MANURE WASTEWATER...........................73

Introduction........................................................................................................... 73
Virus Inactivation during Anaerobic Digestion ............................................... 73
Viral Attachm ent to Soil................................................................................... 74
Purpose .......................................................................................................................76
M materials and M ethods ............................................................................................ 77
Collection and Analysis of Soil Samples ......................................................... 77
Collection and Analysis of Wastewater, Groundwater, and Rainwater
Samples......................................................................................................... 77
Viruses and Viral Assays.................................................................................. 77
Virus Stability................................................................................................... 78
Attachm ent and Detachm ent Studies................................................................ 78
Attachm ent and Detachm ent M echanism s ....................................................... 80
Virus Survival in Soil ....................................................................................... 82
Risk Assessm ent of Flushed Dairy M anure ..................................................... 82
Statistical Analysis ...........................................................................................82
Results.........................................................................................................................83
Soil Characteristics........................................................................................... 83
Batch Studies.................................................................................................... 83
Column Studies ................................................................................................ 84
Conductivity and pH ......................................................................................... 84
Attachm ent and Detachm ent M echanism s ....................................................... 85
Risk Assessment of Viruses Following Land Application...............................90
Survival of Viruses in Soil ............................................................................... 90
Discussion and Conclusions .................................................................................... 90

7 SUM M ARY OF CONCLUSIONS......................................................................... 122

LIST OF REFERENCES............................................................................................... 125

BIOGRAPHICAL SKETCH ........................................................................................1... 40















LIST OF TABLES


Table Raeg

3-1. Percentage of carbon from various sources in yeast extract...................................49

3-2. Characterization of samples used for all experiments............................................50

3-3. Growth of indicator and pathogenic bacteria in the soluble fraction of
w astew after ........................................................................................................... 51

3-4. Growth of indigenous indicator bacteria and S. aureus in the whole fraction of
w astew after. ........................................................................................................... 52

3-5. Comparison of growth between indigenous bacteria in wastewater at 38C and
280C ..........................................................................................................................53

3-6. Determination of inhibitory or nutritional limitation on the growth of indigenous
S. aureus. ..................................................................................................................54

3-7. Growth of S. aureus in soluble effluent and soluble effluent with various
am endm ents........................................................................................................... 55

5-1. Impact of indigenous microflora on viruses suspended in groundwater and
w astew after. .......................................................................................................... 72

6-1. Characteristics of viruses used in the study............................................................97

6-2. Characteristics of test soil.......................................................................................98

6-3. Adsorption of Viruses in groundwater and wastewater to soil................................99

6-4. Elution of viruses adsorbed to soil. ........................................................................100

6-5. Elution of viruses adsorbed in soil columns..........................................................101

6-6. Conductivity and pH changes in soil during batch and column experiments .........102

6-7. Adsorption of viruses in groundwater and wastewater to soil adjusted to pH 7.0..103

6-8. Influence of pH on adsorption of viruses to soil ...................................................104

6-9. Elution of viruses adsorbed to soil by detergents in groundwater.........................105









6-10. Recovery of viruses with 3% BE from soil pretreated with detergents...............106

6-11. Estimated risk of infection from exposure to groundwater following land
application of w astew ater..................................................................................... 107















LIST OF FIGURES


Figure pMag

4-1. Survival of fecal coliforms and GFP producing E. coli in wastewater............... 62

4-2. Passage of GFP E. coli through the pilot-scale fixed-film anaerobic digester .....63

6-1. Adsorption of viruses in groundwater and wastewater to soil.......................... 108

6-2. Adsorption of viruses in groundwater and wastewater to soil columns ........... 109

6-3. Correlation between soluble chemical oxygen demand retained by the soil
and MS2 detachment from soil columns ........................................................110

6-4. Adsorption of MS2 in fractionated wastewater to soil ......................................111

6-5. Adsorption of PRD in fractionated wastewater to soil ....................................112

6-6. Adsorption of DX174 in fractionated wastewater to soil ..................................113

6-7. Influence of influent compounds smaller than 100 kDa on adsorption of
viruses to soil. .................................................................................................. 114

6-8. Inactivation of MS2 by wastewater compounds smaller than 10 kDa...............115

6-9. Correlation between virus pI and virus adsorption to soil .................................116

6-10. Adsorption of viruses in groundwater with 0.01% final concentration of
detergents to soil. ............................................................................................. 117

6-11. Adsorption of viruses in influent with 0.01% final concentration of detergents
to soil...........................................................................................................1...... 18

6-12. Adsorption of viruses in effluent with 0.01% final concentration of detergents
to soil............................................................................................................ 119

6-13. Adsorption of viruses in groundwater to soil pretreated with detergents ..........120

6-14. Survival of viruses in soil. .................................................................................. 121















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

FIXED-FILM ANAEROBIC DIGESTION: MECHANISMS OF PATHOGEN
REDUCTION AND IMPACTS ON VIRUS ADSORPTION TO SOIL

By

Johnny A. Davis

August 2006

Chair: Samuel R. Farrah
Cochair: Ann C. Wilkie
Major Department: Microbiology and Cell Science

Anaerobic digesters have been used for treatment of various wastewaters. Systems

are usually operated at mesophilic (350C) temperatures and long hydraulic retention times

(HRTs) (> 20 days). Pathogen reduction during anaerobic digestion is mainly correlated

with operating temperature and HRT. Higher temperatures and longer HRTs result in

increased rates of pathogen decimation.

Previous studies show that a novel fixed-film anaerobic digester, operated at a low

HRT (5 3 days) and ambient temperature (<280C) treating flushed dairy manure

wastewater, achieved significant reductions of indicator and pathogenic bacteria. Thus,

we sought to determine factors that contributed to indicator and pathogenic bacteria

reduction during operation at a low HRT and ambient temperature. The presence of

indigenous microflora was found to reduce the proliferation of indicator and pathogenic

bacteria. Bacteriophages were also inactivated by the presence of indigenous microflora.

Also, decimation of Staphylococcus aureus was attributed to starvation, resulting from









anaerobic digestion, while not suppressing the other test organisms to the same degree.

Furthermore, attachment to the retained biofilm (i.e., fixed-film) contributes to reduction

by removing indicator and pathogenic bacteria from the liquid phase during anaerobic

digestion.

Next, our study examined the impact of anaerobic digestion on virus adsorption to

soil following land application of treated wastewater. Anaerobic digestion increased

retention of viruses to the soil and decreased mobility of attached viruses through the soil

matrix, as compared with untreated wastewater. Anaerobic digestion removed

compounds) that interfered with virus adsorption to soil. Initial characterization showed

that these compounds) were less than 100 kDa in size. These compounds) interfered

with hydrophobic and electrostatic interactions used by the viruses for adsorption to soil.

Futhermore, compounds) less than 10 kDa in size caused inactivation of MS2.

Our results show the critical role of indigenous microflora and the retained biofilm

during anaerobic digestion in a fixed-film system. The indigenous microflora and

attachment to the biofilm causes decimation of indicator and pathogenic organisms from

flushed dairy manure wastewater, thereby reducing the environmental load of these

organisms during land application. Anaerobic digestion increases the retention of

residual viruses within the soil matrix, therefore reducing the likelihood of contaminating

groundwater.














CHAPTER 1
INTRODUCTION

Livestock are known reservoirs of pathogenic organisms and these organisms are

shed with feces. Also, animal husbandry generates over 1.6 billion tons of manure per

year in the United States (Altekruse et al., 1997). Pathogenic organisms in manure may

be introduced to the environment by land application. Application of solid manure (as

fertilizer) or diluted manure slurry (for fertirrigation) to land is a common management

strategy used by concentrated animal feeding operations (CAFOs). Although this

practice provides nutrients for crop production, pathogenic organisms may persist in

manure, and potentially introduced to the environment and eventually to the herd and

public (Hill, 2003).

In regards to public health, animal manure is a source of food contamination and

has been linked to outbreaks of foodbome illnesses (Park and Diez-Gonzalez, 2003).

Food can become contaminated with pathogenic organisms associated with manure

during food processing (i.e., slaughter) or by land application (i.e., contamination of

uncooked fresh produce) (Troutt et al., 2001). Therefore, pathogenic organisms

introduced by manure into the food chain can result in outbreaks of foodbome illnesses.

Although viruses and protozoa may also be associated with foodbome illnesses,

bacteria have been found to be the most common cause of outbreaks in the United States.

The Centers for Disease Control and Prevention (CDC) reported 75% (655 of 878) of

foodbome-disease outbreaks from 1993 to 1997 were of bacterial origin (Centers for

Disease Control and Prevention, 2000). Furthermore, the Economic Research Service









(ERS) of the United States Department of Agriculture estimated the annual cost

associated with five bacterial agents of foodborne illnesses to be $6.9 billion.

Effective treatment strategies must be used to reduce the levels of pathogenic

bacteria in manure. However, to provide an incentive to implement new technology on

CAFOs, treatment strategies should not only reduce the level of pathogenic organisms,

but also offer on-farm and environmental benefits. Therefore, anaerobic digestion is a

treatment strategy that can be used to reduce pathogen levels in manure and offer on-farm

benefits such as bioenergy production, odor reduction, and biofertilizer production. A

fixed-film anaerobic system at the University of Florida Dairy Research Unit (DRU) has

been shown to decrease pathogen levels and supply bioenergy and biofertilizer for on-

farm use (Davis et al., 2001, Wilkie, 2000)

The persistence of pathogenic organisms in manure can potentially result in

public health concerns. Current manure management strategies used by CAFOs may be

improved to control the levels of pathogenic bacteria in manure. Improvements should

include implementing a manure treatment technology that offers on-farm and

environmental benefits in addition to controlling pathogenic bacteria associated with

manure. Thus, anaerobic digestion is a viable option that offers pathogen control and on-

farm benefits. The fixed-film anaerobic system used by the DRU has been shown to

offer on-farm benefits while reducing the level of pathogens from manure. However, the

factors) that contribute to pathogen reduction during fixed-film anaerobic digestion and

the effect on pathogen transport through soil following land application of treated

wastewater remains unknown. Therefore the purpose of the current study was to: 1)

determine the factors) that contribute to pathogen reduction during fixed-film anaerobic






3

digestion and 2) determine the effect of fixed-film anaerobic digestion on virus transport

through soil after land application.














CHAPTER 2
PATHOGENIC ORGANISMS IN BOVINE MANURE AND THEIR REDUCTION BY
ANAEROBIC DIGESTION

Pathogenic bacteria that pose herd and human health concerns are associated with

manure (Bicudo and Goyal, 2003, Huston et al., 2002, Wang et al., 1996, Wells et al,

1991). Furthermore, pathogenic bacteria can survive in manure for several days once

voided from the animal (Himathongkham et al., 1999). Therefore, exposure to manure

can potentially cause animal diseases, resulting in lost revenues for the animal operation.

Also, environmental contamination may result in human exposure to zoonotic organisms,

which may lead to potential diseases. Environmental contamination and transmission of

zoonotic organisms to humans from livestock manure has been the focus of recent

legislation to improve manure handling and treatment strategies (U.S. Environmental

Protection Agency, 2003). Another concern with manure is the emergence of antibiotic

resistant strains of pathogenic bacteria. The emergence of antibiotic resistant pathogenic

bacteria from manure of antibiotic-fed animals can pose severe herd and human health

issues (Dargatz, 1998). Therefore, the threat of antibiotic resistant pathogenic bacteria

requires that better and more effective pathogen reducing manure management strategies

be implemented by animal operations.

Pathogenic Organisms in Dairy Cattle Manure

Several types of pathogenic bacteria have been isolated from animal manure (Pell,

1997): E. coli 0157:H7, Salmonella spp., Listeria monocytogenes, and Mycobacterium

paratuberculosis. Pathogenic viruses and protozoan have also been isolated from animal






5


manure (Chang et al., 1997, Olson et al., 2004, Valle et al., 1999). Some organisms are

shed in high concentrations when the animal displays illness or may be shed in feces

where the animal does not exhibit any clinical signs of disease. In either case, manure is

a source of pathogenic organisms that may cause either human or herd diseases.

Zoonotic Bacteria

Escherichia coli 0157:H7. Escherichia coli 0157:H7 is an enterohemorrhagic

strain of E. coli, which produces a Shiga-like toxin known as verocytotoxin. The

verocytotoxin is a causative agent of several diseases such as, hemolytic uremic

syndrome (HUS), thrombocytopenic purpura, and hemorrhagic colitis (Pell, 1997,

Prescott et al., 1999, Wells et al., 1991). This organism is considered an emerging

pathogen and has been isolated from raw beef and milk products (Callaway et al., 2003).

Dairy cattle serve as a reservoir of E. coli 0157:H7, where younger animals harbor

the organism more so than mature animals. Wells et al. (1991) measured E. coli

0157:H7 concentrations in fecal samples from cattle in herds associated with two

sporadic cases of hemolytic uremic syndrome and an outbreak of gastroenteritis. The

investigation revealed that 2.3% of calves (5 of 210), 3.0% of heifers (12 of 394), and

0.15% of adult cows (1 of 662) shed E. coli 0157:H7 in feces. Doyle et al. (1997) also

found that E. coli 0157:H7 is isolated predominantly from young animals, with the

highest rate of isolation from postweaned calves. Zhao et al. (1995) found that E. coli

0157:H7 was shed more frequently by weaned calves (4.9 to 5.3%) than preweaned

calves (1.5 to 2.9%). These results show that not only are dairy cattle reservoirs of E.

coli 0157:H7, but younger animals tend to harbor the organism more so than adult

animals.









Escherichia coli 0157:H7 can not only be shed in feces, but also proliferate and

persist in feces once introduced into the environment. Wang et al. (1996) showed that E.

coli 0157:H7 could survive and proliferate in bovine feces up to 21 days at 370C, 49 to

56 days at 220C, and 63 to 70 days at 50C, although no growth occurred at 5C.

Nicholson et al. (2005) found that E. coli 0157:H7 could survive for 93 days in dairy

manure slurry stored at less than 200C. However, survival decreased to 32 days

following land application without storage. Park and Diez-Gonzalez (2003) found that E.

coli 0157:H7 could survive in cattle manure for at least 25 days at room temperature.

Salmonella spp. Salmonella spp. is the most common pathogen associated with

livestock manure (Stehman, et al., 1996). These organisms are facultative anaerobic,

gram-negative rods. These organisms are non-species specific (e.g., equine, swine,

poultry, and bovine), can be transferred from animals to humans through undercooked

meat or raw eggs, and are causative agents of typhoid fever, enteric fevers, septicemia,

and gastroenteritis. Salmonella spp. causes gastroenteritis in humans and can be fatal to

young, elderly, or immuno-compromised individuals (Dargatz, 1998). From 1993 to

1997, 55% of bacterial foodborne-disease outbreaks in the United States were caused by

Salmonella spp (Centers for Disease Control and Prevention, 2000). The Economic

Research Service (ERS) estimated annual economic loss due to foodborne Salmonella

infections in 1996 and 2003 was $3.5 and $2.9 billion, respectively (Buzby et al., 1996,

ERS, 2003, Frenzen et al., 1999).

The emergence of antimicrobial resistant Salmonella spp. has become a major

health concern. In particular, S. typhimurium DT104 has developed resistance to various

antibiotics including ampicillin, sulfonamides, tetracycline, chloramphenicol, and









streptomycin (Dargatz, 1998). Park and Diez-Gonzalez (2003) found that S. typhimurium

DT104 could survive in cattle manure for at least 25 days at room temperature. During

slaughter, meat can potentially become contaminated with S. typhimurium DT104.

Galland et al. (2001) identified 59 isolates as S. typhimurium in a survey of five slaughter

establishments processing cull (market) dairy cattle. Out of the 59 isolates, 35 were

obtained from one facility, where 88.6% of those isolates were confirmed as S.

typhimurium DT104.

Salmonella spp. are commonly associated with dairy cattle. Huston et al. (2002)

found that out of 2,283 individual fecal samples and 15 bulk milk tank samples from five

dairy herds, 11% of calves, 56% of mature cows, and 20% of bulk tank samples were

positive for Salmonella spp. The study also found that mature cows were 21 times more

likely to be shedding Salmonella spp. than were unweaned calves. Furthemore, no

clinical signs of salmonellosis were identified in any of the adult animals in the five

herds. The study concluded that Salmonella spp. can be shed in feces of mature healthy

cows, however bacterial concentrations were not determined during the study.

Dairy cattle can also serve as a vehicle for Salmonella spp. contamination of meat.

Troutt et al. (2001) measured prevalence of Salmonella spp. in five beef slaughter

establishments in five different regions of the United States. The investigators found a

23.1% overall prevalence of Salmonella spp. Additionally, the highest prevalence

(54.5%) and the lowest (4.3%) prevalence were found during the summer sampling

period and in two different locations. The investigators were able to detect Salmonella

spp. in all the establishments surveyed. These results suggest that the prevalence of

Salmonella spp. is not dependant on seasonal variations nor is the organism isolated to









one region of the country. Galland et al. (2001) determined Salmonella serotype

diversity and prevalence from the same samples obtained in the study by Troutt et al.

(2001). The investigators identified 58 serotypes of Salmonella, where Salmonella ser.

Montevideo was the most frequent (21%). Other serotypes (Muenster, Kentucky,

Mbandaka, Senftenberg, and Typhimurium) were found in all establishments surveyed.

Campylobacter spp. Campylobacter spp. are microaerophilic, gram-negative

vibriod cells. They are usually found in the oral cavity, reproductive organs, and

intestinal tract of animals and humans. Two species of particular concern are C. jejuni

and C coli. These organisms cause human enteritis and are associated with livestock.

Campylobacter spp. may enter the supply of raw milk or water from bovine feces

(Wesley et al., 2000). Healthy cattle are potential reservoirs of C. jejuni and C coli and

the incidence of disease is seasonal (Wesley et al., 2000).

Mycobacterium paratuberculosis. Mycobacterium paratuberculosis is an aerobic,

acid-fast, rod-shaped organism. This slow-growing, fastidious organism generally

requires the presence of mycobactin, a growth factor that serves as an iron chelator, to

proliferate (Grant et al., 1996). Mycobacterium paratuberculosis is the causative agent

for Paratuberculosis (PTB), or Johne's disease, in cattle and has been implicated in

Crohn's disease in humans (Grant et al., 1996). Paratuberculosis is a chronic,

granulomatous intestinal disease that is manifested by nonresponsive diarrhea,

progressive weight loss, and death. Mycobacterium paratuberculosis infections are not

confined to specific tissues and the organism can be isolated from several organs (Hines

et al., 1987). This organism has also been isolated from uterine flush fluids of infected

dairy cattle (Rohde et al., 1990).









Several techniques have been developed to detect M. paratuberculosis. Collins et

al. (1990) described the use of radiometry for detection. A study of nine infected dairy

herds in Wisconsin showed that radiometric detection using filter-concentrated manure

samples detected 92% of M. paratuberculosis from feces, while conventional culture

techniques using HEY agar only detected 60%. Molecular detection of M.

paratuberculosis has also been studied. Millar et al. (1996) found that IS900, an unusual

DNA insertion element unique to M. paratuberculosis, can be used to detect this

organism in pasteurized milk.

Paratuberculosis has an adverse effect on milk production (Hernandez and Baca,

1998). Mycobacterium paratuberculosis can be shed in milk in high concentrations

(Sweeney et al., 1992) and can survive pasteurization temperatures (Grant et al., 1996).

This organism can also be transmitted in utero (Seitz et al., 1989) and by herd-to-herd

contact (Collins et al., 1994). The prevalence of this organism at a dairy can be attributed

to environmental conditions, manure-handling practices, newborn calf care, and grower

calf care (Goodger et al., 1996).

Listeria monocytogenes. Listeria monocytogenes are facultative anaerobic, gram-

positive, rod-shaped organisms. These organisms cause listeriosis in animals and humans

(George et al., 1996, Jensen et al., 1996, Mussa, et al., 1999). Jensen et al. (1996) found

that L. monocytogenes isolated from humans and cows were of the same serotype. This

implies that milk contaminated with this organism can possibly infect humans.

The ability of this organism to survive in food causes concern for human health

(George et al., 1996). Gohil et al. (1996) found that L. monocytogenes can survive up to

three days in labneh and houmos, two traditional Arabic foods. Listeria monocytogenes









have also been shown to survive in acidic foods (e.g., yogurt, cottage, mozzarella, and

cheddar cheeses) by induction of the acid tolerance response (ATR) (Gahan et al., 1996).

The ATR mechanism involves the synthesis of proteins, including outer membrane and

heat shock proteins, which provide a mechanism for maintaining intracellular

homeostasis in suboptimal, or lethal, environments.

Another concern for human health is that antibiotic resistant strains of these

organisms have been detected. Erythromycin resistant (Em') and tetracycline resistant

(Tet) Listeria spp. has been isolated from food (Roberts et al., 1996). The use of high

pressure (600 MPa) has been studied as a possible treatment process for food. Mussa et

al. (1999) found that the use of high pressure in the pasteurization reduced the

concentration of L. monocytogenes in milk.

Mastitic Bacteria

Staphylococcus aureus. Staphylococcus aureus are gram-positive, facultative

anaerobic cocci. These organisms are common inhabitants of the skin and mucous

membranes of warm-blooded animals. Staphylococcus aureus are causative agents of

peracute, acute, chronic, subclinical mastitis in dairy cattle and can be excreted in milk

(Umeki et al., 1993). Subclinical infections are the most common form of mastitis

caused by S. aureus, however this organism can also cause clinical mastitis (Elbers et al.,

1998). Staphylococcus aureus induced mastitis (i.e., contagious mastitis) is characterized

by invasion of the mammary gland tissues, where the mammary gland parenchyma is

replaced by granulation tissue, fibrosis occurs, and multiple abscesses form (Cullor,

1993). Although antibiotic therapy has been used to treat infections, resistant strains of S.

aureus associated with mastitis have emerged in recent years (Malinowski and

Klossowska, 2003).









Streptococcus spp. Streptococci are facultatively anaerobic gram-positive cocci.

These organisms have a fermentative metabolism where lactate is the primary product.

These organisms are common inhabitants of the mouth and upper respiratory tract of

vertebrates. Several species of streptococci are causative agents of bovine mastitis. A

streptococcus responsible for contagious mastitis is S. agalactiae. Streptococcus uberis

and S. dysgalactia are associated with environmental mastitis.

Contagious mastitis infections caused by S. agalactiae (Lancefield group B) can be

either clinical or subclinical. Infections involve adherence of the organism to mammary

gland cells. Although, Lammers et al. (2001) showed poor adhesion to mammary gland

cells by S. agalactiae, fibrinogen-binding proteins secreted by the organism have been

shown to aid in adhesion to mammary gland cells (Jacobsson, 2003).

A single strain of S. agalactiae has been shown to be the causative agent of

mastitis. Merl et al. (2003) studied 79 streptococcal isolates from subclinical mastitis of

54 cows. The study found that a single strain of S. agalactiae were responsible for all the

infections. In addition to mastitis, S. agalactiae can cause severe invasive disease in

humans, especially in neonates (Bohnsack et al., 2004). Evidence suggests that S.

agalactiae may be a zoonose, being transmitted from cows to humans and visa versa,

although the risk of transmission between species is considered low (Sukhnanand et al.,

2005).

Environmental mastitis infections may be related to environmental factors such as

insect population (Yeoman and Warren, 1984) and bedding materials (Hogan et al.,

1990). "Summer mastitis" is a teat infection caused by several organisms including S.

dysgalactiae and is spread by a fly, Hydrotaea irritans (Yeoman and Warren, 1984).









Summer mastitis infections caused by S. dysgalactiae were found to be five times more

prevalent in herds without fly control versus herds with fly control (Nickerson et al.,

1995).

The type of material used for bedding can result in increased herd exposure to

environmental mastitic streptococci. Care and type of bedding material (i.e., organic or

inorganic) used in freestall barns have a direct impact on exposure to environmental

mastitic streptococci (Bey et al., 2002). Hogan et al. (1990) found that environmental

mastitic streptococci were able to proliferate in organic bedding material and increase the

concentration of these organisms on teats. Zehner et al. (1986) found S. uberis was able

to proliferate in various types of organic bedding (e.g., recycled manure, chopped straw,

hardwood chips, paper, and softwood sawdust). Additionally, Todhunter et al. (1995)

reported streptococci concentrations of 6 to 7 logo CFU/g dry weight of various bedding

materials (i.e., pelleted corn cobs, recycled manure, and wood shavings) throughout the

year. Although streptococci concentrations did not vary with seasonal conditions, the

rate of environmental streptococcal mastitis (reported as intramammary infections per

cow per day) was highest during the summer and lowest during the winter. These results

suggest that the use of organic bedding material can lead to environmental mastitis by

providing conditions for mastitic streptococci to survive and proliferate.

Pathogenic Viruses

BVDV. Bovine virus diarrhea is an infectious disease in cattle caused by a

retrovirus, BVDV. BVDV is a member of the Pestivirus genus, within the Flaviviridae

family. The attachment and entry of flavivirus is mediated by the envelope (E) protein

(-50 kDa), a major glycoprotein on the virus particle (Lee and Lobigs, 2000). BVDV is

found in oculonasal discharges and is transmitted by close and direct contact within the









herd. An important feature of this disease is that an infected pregnant female can give

birth to a persistently infected calf that will excrete BVDV throughout its life (Valle et

al., 1999). Valle et al. (2000) studied BVDV sero-conversion (a surrogate measure for

incidence) in Norwegian dairy herds and found that there was a steady decline in sero-

conversion risk. Although sero-conversion continued over time, the decrease was

attributed to effective control of major risk factors. These factors include the use of

common pastures, purchasing animals associated with BVDV, and herd-to-herd contact

over pasture fences (Valle et al., 1999).

Bovine rotavirus. Bovine rotavirus is a member of the Reoviridae family, has a

genome that consists of 11 segments of dsRNA, and is enclosed by a triple-layered

capsid. Rotaviruses are a common cause of diarrhea in calves and humans (Chang et al.,

1997, Klingenberg et al., 1999). There are three groups of rotaviruses: A, B, and C.

Group A is a common cause of neonatal calf enteritis (Klingenberg et al., 1999). Group

B has been implicated in sporadic cases of diarrhea in calves (Chang et al., 1997). Group

C has been suggested as a common enteric pathogen in animals and humans, but are

difficult to cultivate because they are fastidious in their in vitro cell culture requirements

(Alfieri et al., 1999).

Activation of rotavirus infectivity requires the presence of exogenous proteases,

which cleave the VP4 protein. Replication is completely cytoplasmic, where rotaviruses

supply necessary enzymes to replicate dsRNA. Subviral particles form and maturation

occurs through budding of the endoplasmic reticulum (ER). Finally, viral particles are

released through cell lysis.









Two major proteins, VP4 and VP7, on the outer shell of rotaviruses have been

studied extensively (Alfieri et al., 1999, Chang et al., 1997, Klingenberg et al., 1999).

The genes of these two outer capsid proteins are used for molecular detection of

rotaviruses using reverse transcription polymerase chain reaction (RT-PCR). Buesa et al.

(1996) found that RT-PCR is highly effective for G (VP7 gene) and P (VP4 gene)

genotyping of rotaviruses, but not substantially more sensitive than enzyme linked

immunosorbant assay (ELISA) and electron microscopy (EM) for detection in fecal and

environmental samples. To control the spread of this pathogen, preventive measures

should be aimed at better hygiene, herd management, and vaccination programs

(Klingenberg et al., 1999).

Parasitic Protozoa

Cryptosporidia. Cryptosporidia are protozoan parasites that are commonly

associated with human and livestock manure (Olson et al., 2004). The organism exists as

an oocyst during the infectious stage. Oocyst are biologically dormant, easily transported

in water and can survive drinking water treatment (i.e., chlorination). Although there are

several species of cyptosporidia (e.g., C. andersoni and C. muris), only C parvum is

clinically significant. Cryptosporidium parvum is the causative agent of

cryptosporidiosis in humans, which is characterized by severe diarrhea and dehydration.

Cattle are considered the major reservoir of C. parvum (Olson et al., 2004).

Younger calves are more susceptible to infection and are more likely to shed oocysts

during infection than are older animals. Sischo et al (2000) measured the prevalence of

cryptosporidia in dairy cattle and calves. The investigators detected Cryptosporidium in

10 of 11 farms surveyed. Furthermore, calves (0 to 3 weeks old) had the highest

prevalence of cryptosporidia infections among older calves (> 4 weeks old) and mature









cows. Additionally, the investigators evaluated several risk factors for spreading

crytosporidia among calves. Contact between calves and frequent bedding changes (> 12

times per year) were found to be the two main management risk factors associated with

fecal shedding of cryptosporida. Younger calves are more likely to become infected and

shed oocysts. However, adult animals are can shed oocysts where infections are

asymptomatic (Graczyk et al., 2000).

The field spreading of contaminated calf manure can potentially introduce oocysts

to the watersheds (Walker and Stedinger 1999). To contaminate watersheds, oocysts

must transported by manure and released into a watershed. Schijven et al. (2004)

measured oocyst release from manure by water application either as a mist or droplets.

Calf and cow manure were used separately or mixed together in the study to simulate

manure management practices at the dairy operation. The study found that oocysts were

more readily released from calf manure than cow manure. The larger coarse particles

(i.e., undigested hay or grain) in cow manure do not readily partition into the aqueous

phase. However, calf manure consisted of finer colloidal particles, which partitioned

easily into the aqueous phase. The differences in manure composition are directly

impacted by the difference in diet among cows and calves. The study also found that

droplet size and water application rate was directly proportional to oocyst release rates.

This result suggests that during heavy rainfall events, oocysts can be released from

manure and potentially be transported in runoff.

Anaerobic Digestion and Pathogenic Organisms

Anaerobic Digestion

Anaerobic digestion is the natural process of converting complex organic

compounds to methane and carbon dioxide by a series of symbiotic microbial interactions









in the absence of oxygen. The process of anaerobic digestion is utilized as a means of

degrading organic matter in the environment, providing nutrition for ruminants, and

treating different industrial and agricultural organic waste products. Ruminant organisms

have evolved to utilize anaerobic digestion for degrading and deriving energy from

otherwise indigestible plant material. Anaerobic degradation under controlled conditions

provides an effective method for treating organic wastes. The development of enclosed

systems (i.e., anaerobic digesters) has enhanced the process of anaerobic digestion by

providing an optimum environment for methanogenesis.

Anaerobic Digesters

Anaerobic digesters are bioreactors designed to optimize the process of anaerobic

degradation by providing an environment under controlled conditions to facilitate the

growth of the anaerobic bacteria. These systems are designed for wastewater

stabilization and energy production (i.e., methanogenesis). Other benefits derived from

anaerobic digesters are odor reduction (Powers, 1999) and bacterial pathogen decimation

(Kearney, 1993). Several types of digesters have been studied for treating manure.

These include anaerobic lagoons, continuous stirred tank reactors, plug-flow digesters,

and fixed-bed reactors.

Anaerobic Lagoons. Preceding digesters, anaerobic lagoons have been used to

treat animal manures. However, lagoons do not offer biogas recovery for energy

production. Development of covers over lagoon systems permitted biogas recovery for

energy use. These systems are operated at ambient temperature, resulting in seasonal

changes in biogas production. Covered anaerobic lagoons require hydraulic retention

times (HRTs) in excess of 40 days. These systems have been found to achieve COD

reductions of 70% to 94% and produce biogas with various methane contents (70% to









88% CH4) (Cheng et al., 1999, Safley and Westerman, 1992, Williams and Frederick,

2001).

Continuous Stirred Tank Reactors. Continuous stirred tank reactors (CSTRs)

are insulated, cylindrical tanks that are either continuously or intermittently fed, where

the volume of effluent equals the volume of influent. These systems are used for

methane recovery from livestock manure that usually contains a 5% to 12% total solids

concentration. The contents of CSTRs are mixed either by mechanical methods or

recycling biogas through the liquid. These systems are operated at HRTs of 10 to 20

days (Wilkie and Colleran, 1989) and maintained at either mesophilic (-350C) or

thermophilic (-550C) temperatures, although psychrophilic (5C to 20C) operating

temperatures have been studied (Zeeman et al., 1988).

Plug-flow Digesters. Plug-flow digesters are systems used to treat scraped manure

with an 11% to 14% total solids concentration (Hills, 1983, Hills and Mehlschau, 1984,

Liu, 1998). They are usually long rectangular tanks, often built into the ground, and

equipped with an impermeable plastic cover for biogas recovery. Manure is not mixed as

it moves through the system as a combined mass or "plug". Plug flow systems usually

operate at HRTs in excess of 30 days.

Fixed-bed Reactors. Fixed-bed anaerobic reactors use a support medium to retain

anaerobic organisms. A fixed-bed system is constructed with an internal support media

to provide surface area for the attachment and retention of anaerobic bacteria as a

biofilm. This advancement allows for the retention of anaerobic bacteria within the

digester independent of HRT (Wilkie, 2000). Adhesion of anaerobic bacteria to inert

support media, facilitating biofilm formation, is influenced by bacterial surface properties









and support media characteristics (Verrier et al., 1987). The surface texture and porosity

of the support media have a significant impact on system performance, where open-pored

surface texture and high porosity results in higher biofilm efficiency (Show, 1999). A

major advantage of fixed-bed technology is the immobilization of methanogens, which

grow at a slow rate (Wilkie and Colleran, 1989). Fixed-bed technology allows for the

bacteria to be retained within the digester, resulting in a decrease in HRT needed to digest

wastewater (Wilkie, 2000). This type of wastewater management system is capable of

treating larger volumes of dilute wastewater per unit time than conventional systems

(Wilkie, 2000). Fixed-bed anaerobic reactors are suited for treating slurry with low (<

1%) total solids concentrations.

Microbial Diversity in Anaerobic Digesters

The microbial ecosystem within anaerobic digesters is primarily a consortium of

bacteria that are involved in synergistic interactions for the digestion of organic

compounds for biogas production (Bitton, 1994, Wilkie and Colleran, 1987). The

process of digestion, involves four groups of bacteria: 1) hydrolytic bacteria, 2)

acidogenic bacteria, 3) acetogenic bacteria, and 4) methanogenic bacteria. The four

groups of bacteria are dependent on each other for maintaining an optimum environment

for anaerobic digestion.

Hydrolytic Bacteria. Hydrolytic bacteria degrade complex organic material (e.g.,

polysaccharides, proteins, lipids), yielding monomers that are subsequently utilized by

the acidogenic bacteria. Hydrolytic species inhabiting digesters feed ruminant manure

originate from organisms that survive passage from the rumen and into feces. Velazquez

et al. (2004) identified a novel species ofPaenibacillus, P. favisporus sp. nov. This









organism was isolated from bovine feces and found to produce a variety of hydrolyic

enzymes, including xylanases, cellulases, and amylases.

Acidogenic Bacteria. The acidogenic bacteria convert the monomers (e.g.,

monosaccharides, fatty acids, amino acids) to organic acids, alcohols, and ketones.

Acidogenic bacteria include Propionium spp. that produces propionate and Butyrvibirio

spp. Butyrvibiriofibrisolvens are gram-negative, non-sporeforming, curved rods. These

organisms mainly utilize mono- and disaccharides as substrates, resulting in the

production of butyric acid (Bryant and Small, 1956).

Acetogenic Bacteria. The acetogenic bacteria primarily utilize propionate and

butyrate as substrates within anaerobic digesters. Syntrobacter wolinii utilizes propionate

and Syntrophomonas wolfei utilizes butyrate, where both organisms form acetate, CO2,

and H2 (Wilkie and Colleran, 1987).

Methanogenic Bacteria. There are two primary groups of methanogenic bacteria

active in anaerobic digesters: 1) hydrogen-utilizing methanogens and 2) acetate-utilizing

methanogens. Hydrogenotrophic methanogens utilize CO2, and H2 for methane

production. These organisms are not primary methane producers in the digesters,

however their activity maintains a low partial pressure of hydrogen to drive interspecies

hydrogen transfer within digesters. Acetotrophic methanogens (e.g., Methanosarcina

barkeri and Methanothrix soehngenii) utilize acetate to form methane (CH4).

Methanosarcina barkeri is usually found in systems operating at low HRTs, whereas

Methanothrix soehngenii is found in systems operating at high HRTs (Wilkie and

Colleran, 1987). Acetate is the primary source of methane and accounts for









approximately 70% of the methane produced within digesters (Wilkie and Colleran,

1987).

Pathogen Decimation during Anaerobic Digestion of Animal Manure

Anaerobic digestion for stabilizing manure offers a non-chemical solution to

control pathogenic organisms. Anaerobic digesters provide a controlled and optimum

environment for the natural process of anaerobic degradation. The use of anaerobic

technology to control pathogenic organisms has been studied for several systems. The

survival of pathogenic organisms during anaerobic digestion of animal manure is

influenced by several factors. Operating temperature and HRT have been shown to

influence the rate of pathogen decimation during anaerobic digestion. However,

environmental conditions during anaerobic digestion may also contribute to the rate of

pathogen decimation. The pH of digester contents affects pathogen survival during

digestion and systems have been designed to use this mechanism to reduce pathogen

concentrations (Huyard et al., 2000). Substrate availability, microbial competition for

substrates, and bacterial production of antibacterial compounds may also contribute to

decimation of pathogenic organisms during anaerobic digestion (Kearney et al., 1993,

Sahlstrom, 2003, Smith et al., 2005).

Temperature. One of the most important parameters influencing the rate of

reduction of pathogenic organisms during anaerobic digestion is operating temperature.

Temperature influences the kinetics of anaerobic digestion, which affects environmental

conditions of digester contents. Anaerobic digesters are usually operated at either

mesophilic (-350C) or thermophilic (-550C) temperatures.

Mesophilic operation maintains the digester at or near the temperature pathogens

are exposed to in the animal. However, anaerobic digestion at this temperature range has









been shown to achieve significant reductions of pathogenic organisms. Duarte et al.

(1992) demonstrated that mesophilic anaerobic digestion with a 15-day HRT resulted in a

3-logio reduction of total coliforms, fecal coliforms, and streptococci from swine manure.

Thermophilic operations maintain digester temperature in excess of that found within the

animal. The higher operating temperatures have been shown to produce significant

reductions of pathogens in shorter amounts of time as compared to mesophilic digestion

(De Leon and Jenkins, 2002). Decimation times of pathogenic organisms are in the range

of a few hours during thermophilic digestion, whereas mesophilic operations require

several days (Berg and Berman, 1980, Burtscher et al., 1998, Watanabe et al., 1997).

Chauret et al. (1999) found that mesophilic digestion of sewage sludge achieved a 0.3

logo0 reduction of cyptosporida oocysts after 20 days. However, Kato et al. (2003) found

thermophilic digestion of biosolids resulted in > 2 logo reduction of cryptosporidia

oocysts after 1 h.

Although thermophilic operation yields higher pathogen reductions, process

stability has been cited as a disadvantage (Kim et al., 2002). Thermophilic operation

results in rapid volatile acid production from fast-growing thermotolerant acidogens. The

rapid production of volatile fatty acids lowers the pH of the medium, inhibiting the

growth of methanogens, which grow at a slower rate. The low pH encountered in

thermophilic operations reduces concentrations of pathogenic organisms.

Hydraulic Retention Time. The HRT of a digester greatly influences pathogen

survival during anaerobic digestion. The HRT is the average amount of time a desired

substrate remains in the system and is a function of system capacity and feed rate. The

longer the HRT, the longer substrate is retained in the system, which lowers the amount









of substrate that can be treated per unit of time. Systems that operate at long HRTs (>10

days) can achieve over 2 logo reductions of pathogenic organisms from animal manure

(Duarte et al., 1992). Pathogen survival is influenced by HRT, where retention in a

suboptimum environment results in decimation. During the process of anaerobic

digestion, environmental conditions become stressed due to lack of available nutrients

and pathogens cannot maintain a high population density as compared to concentrations

inside the animal.

Influence of Temperature and HRT. The combination of operating temperature

and hydraulic retention time are regarded as the main factors impacting pathogen survival

during anaerobic digestion. Mesophilic operations with long hydraulic retention times

and thermophilic operations with short hydraulic retention times can reduce pathogen

levels from animal manure. However, pathogen reduction during anaerobic digestion is

not based solely on operating temperature and hydraulic retention time (Hill, 2003).

Operating temperature and hydraulic retention time may influence the alteration of the

physico-chemical properties of wastewater during digestion. Alteration of the physico-

chemical properties may have direct impacts on pathogen acclimation and survival in an

anaerobic digester environment.

Volatile Fatty Acids. Short-chain volatile fatty acids (e.g., acetate, propionate, and

isobutyrate) produced by acidogens during anaerobic digestion can contribute to

pathogen decimation. Abdul and Lloyd (1985) demonstrated growth inhibition of E. coli

by various short-chain fatty acids. Cells were grown in 0.1% glucose under anaerobic

conditions. Short-chain volatile fatty acids were added to the medium as potassium salts

during early log phase. The authors demonstrated inhibition of growth by acetate (60 and









120 mM), propionate (52 and 104 mM), and isobutyrate (60, 90, and 180 mM). Kunte et

al. (1998) demonstrated inactivation of S. typhi in cattle manure by volatile fatty acids

during anaerobic digestion for 30 days (control) and 15 days (experimental). The feed

for the experimental digester was supplemented with 1% glucose daily. The decimation

times (To9), the time required to achieve a 1 logo reduction, were significantly lower for

experimental digesters producing high volatile fatty acid concentrations (5 g/L) as

compared to control digesters producing lower volatile fatty acid concentrations (0.1

g/L). However, the authors did not report operating temperature of each digester. Kwon

and Ricke (1999) demonstrated growth inhibition of Salmonella typhimurium by

propionic acid and sodium propionate. Cells were grown in tryptic soy broth (TSB) with

various concentrations of propionic acid or sodium propionate. The culture medium was

adjusted to pH 7.0. Inhibition of S. typhimurium was observed with propionic acid and

sodium propionate concentration > 25 mM.

Effect of pH. The pH of digester contents has significant effects on pathogen

survival during anaerobic digestion. The production of short-chain volatile fatty acids

during anaerobic digestion lowers the pH of the medium (Munch et al., 1998). Short-

chain volatile fatty acids can contribute to pathogen decimation by lowering

environmental pH. However, during anaerobic digestion, short-chain volatile fatty acids

serve as substrates for subsequent methanogenesis. In a single-phase digester, where

acidiogenesis and methanogenesis occur in the same reactor, the process of

methanogenesis counteracts the pH effects of volatile fatty acids with the production of

bicarbonate and the consumption of acetate. Bicarbonate buffers the digester contents,

maintaining a pH of 6.5 to 7.0. Thus, pH may not contribute to pathogen decimation









during single-phase anaerobic digestion. Two-phase anaerobic digesters have been

developed that separate the acidogenic phase from the methanogenic phase of anaerobic

digestion. The acidogenic phase is usually operated at theromophilic (>550C)

temperatures (Yu and Fang, 2000) and short HRTs (Huyard et al., 2000), resulting in

rapid growth ofthermotolerant acidogenic organisms. The effluent from the acidogenic

phase is fed to the methanogenic phase, which is operated at mesophilic (-35C) and long

HRTs (> 10 days) to accommodate the slow growth rate of methanogens (Huyard et al.,

2000). During the methanogenic phase, short-chain volatile fatty acids are converted to

acetate, hydrogen, and carbon dioxide. These products serve as substrates for

acetotrophic and hydrogenotrophic methanogens. Acidogenic and methanogenic phases

can be operated and controlled independently, allowing for optimization of each phase

without adverse affects to methanogenic and acidogenic populations (Ince, 1998). Phase

separation allows for pathogen reduction during the acidogenic process due to lower pH

resulting from the production of short-chain volatile fatty acids.

Solids Concentration. The survival of pathogens during anaerobic digestion of

manure is influenced by the amount of solids present in digester contents. Pathogens can

become readily associated with organic particles in the environment (Grossart et al.,

2003). Nutrients absorb onto the surfaces of organic particles where pathogens can

adhere and use the solids as a food source (Goulder, 1977). Bacterial adherence may be

achieved by the use of fimbriae (Galfi et al., 1998), S-layers (Kotiranta et al., 1998), and

pili (Omdorff et al., 2004). London-van der Waals forces, hydrophobic, and electrostatic

interactions are the main mechanisms for bacterial and viral adhesion to solids (Ash,

1979, Daniels, 1980). In addition, exopolymeric substances and cell surface proteins also









contribute to adhesion to particles (Wicken, 1985). During anaerobic digestion,

pathogens can attach to organic particles, supplying the organism with a nutrient source,

increasing the likelihood of survival. Kumar et al. (1999) found decreased pathogen

reductions from cattle dung using anaerobic digestion at mesophilic temperature with

increased loading of volatile suspended solids concentrations.

Substrate Availability. The availability of substrate, as organic carbon, has a

significant role in pathogen survival during anaerobic digestion. The amount of organic

carbon available for biological degradation and assimilation is represented by

biochemical oxygen demand (BOD). Anaerobic digesters can significantly reduce BOD

concentrations in animal manure (Chin and Ong, 1993). The reduction in BOD creates a

substrate-limiting environment, and pathogens are not readily suited to such stressed

conditions. Pathogens associated with manure are voided from the intestines of the

animal, an optimum environment. Once voided from the animal, pathogens in manure

are exposed to environmental conditions. As manure enters an anaerobic digester, the

amount of available substrate may fluctuate and pathogens have to be capable of adapting

to changes in substrate fluctuations. The organic material that is available for biological

degradation is mainly used for the formation of methane. During optimized anaerobic

digestion, there is little available substrate to maintain pathogen concentrations found in

the animal (Kearney et al., 1994).

Microbial Competition. Anaerobic digesters are optimum environments for

methanogenesis and are operated for supporting the proliferation of organisms involved

with the process of anaerobic digestion. Therefore, organisms involved with carbon flow

through anaerobic digestion readily degrade and assimilate available organic carbon that









is fed to the system. Affinity for necessary substrates to support proliferation is higher

for organisms involved in the process of anaerobic digestion than for pathogenic

organisms. Saturation constants (Ks) for anaerobic digestion bacteria and substrates are

lower than for other bacteria (Mosche et al., 1998). The lower K, values result in a

higher affinity for substrates for bacteria involved with anaerobic digestion (Song et al.,

2004). Results from the aforementioned studies suggest that pathogens cannot readily

utilize the limited amount of substrate to support a high population as compared with

bacteria associated with anaerobic digestion, resulting in pathogen decimation.

In addition to higher affinity for substrates, differences in concentrations of digester

microflora and pathogens influences microbial competition within anaerobic digesters.

Pathogens are usually found at lower concentrations than nonpathogenic bacteria, unless

voided in high numbers by an infected animal (Huston et al., 2002). Anaerobic digesters

are predominated by bacteria involved with anaerobic digestion and are present in high

concentrations (107 to 108 CFU/ml) (Solera et al., 2001). Pathogens are usually loaded to

the system in lower concentrations as compared to anaerobic digestion bacteria already

present in the system. The difference in population density and the higher affinity for

substrates give anaerobic digestion bacteria a survival advantage in anaerobic digesters,

leaving little opportunity for bacterial indicators and pathogens to become established.

Antimicrobial Products of Bacteria. Bacteria are in constant competition with

other bacterial species for available nutrients in natural environments. Different genera

of bacteria produce substances that have antibacterial properties. The type of substances

produced and the resulting effects can be specific for different species within the same

genus. Bacteriocins and colicins are antimicrobial compounds that are produced by









bacteria to give the organism a competitive advantage in natural environments. The role

of bacteriocins and colicins has not been extensively studied in regards to pathogen

reduction during anaerobic digestion. However, organisms that can be found in animal

manure and subsequently in anaerobic digesters have been shown to produce bacteriocins

and colicins. Holo et al. (2002) reviewed bacteriocins from propionate producing

bacteria that were effective inhibitors of gram positive and gram negative organisms.

Lyon and Olson (1997) isolated colicins produced by Escherichia coli ECL12 (isolated

from bovine feces) that inhibited the growth of pathogenic E. coli strains. Bacteriocins

and colicins may be present and active in digesters, contributing to pathogen decimation

during anaerobic digestion.

Summary

Pathogenic organisms present in animal manure pose a serious herd and human

health concern. Manure management practices have become the focus of recent

legislation to control the transmission of zoonotic organisms to the environment.

Anaerobic digestion has been shown to reduce the level of pathogens from animal

manure. The rate of pathogen decimation during anaerobic digestion has been related to

operating temperatures and hydraulic retention times. However, other factors, such as

pH, volatile fatty acid concentrations, microbial competition, and substrate affinity, have

a significant role in the fate of pathogenic organisms during anaerobic digestion.

Although systems designed for the purpose of pathogen reduction focus on operating

temperature, reactor configurations that can optimize other factors that contribute to

pathogen reduction may prove efficient and superior to current systems for manure

wastewater sanitization.






28

Purpose of Study

The purpose of the current work was to identify the factors) that contribute to the

reduction of indicator and pathogenic bacteria during anaerobic digestion in a fixed-film

system. Also, we sought to study the transport of residual viruses through soil following

land application of anaerobically treated flushed dairy manure wastewater.














CHAPTER 3
INDICATOR AND PATHOGENIC BACTERIA REDUCTION BY ANAEROBIC
DIGESTION: THE ROLE OF MICROBIAL COMPETITION AND SUBSTRATE
LIMITATIONS

Introduction

Several pathogenic bacteria are known to be associated with dairy manure. These

include organisms that mainly infect dairy cattle, such as S. aureus and streptococci, and

zoonotic organims, such as E. coli 0157:H7 and Salmonella spp., which can cause

infections in humans. Therefore, disposal of animal manure by land application raises

environmental and public health concerns. Effective treatment of manure by anaerobic

digestion can be achieved using well-managed systems. Anaerobic digesters have been

shown to achieve 2 to 3 logio reductions of a variety of indicator and pathogenic

organisms from various substrates (DeLeon and Jenkins, 2002, Duarte et al., 1992).

Decimation of pathogenic bacteria during anaerobic digestion may due to inhibitory

effects caused by microbial interactions and competition for limited substrates.

Inhibitory effects of indigenous microflora may involve the production of

secondary metabolites. Such metabolites are produced during stationary phase or

idiophase and usually have antimicrobial properties (Riviere et al., 1975). Secondary

metabolites include antibiotics, colicins and bacteriocins. These compounds are usually

effective against strains related to the producing organism. Lyon and Olson (1997)

studied a bacteriocin, designated colicin ECL 12, from E. coli isolated from swine

manure. Colicin ECL 12 was found to inhibit two strains of E. coli and 17 strains of E.

coli 0157:H7. However, there were no inhibitory effects found against selected gram-









positive pathogenic bacteria (e.g. S. aureus and L. monocytogenes). Bacteriocins may

also demonstrate activity against both gram-positive and gram-negative organisms.

Hyronimus et al. (1998) found that a strain of Bacillus coagulans, isolated from bovine

manure, produced coagulin. Coagulin is a bacteriocin capable of inhibiting a variety of

bacteria including strains of Bacillus spp., Enterococcus spp. and Listeria spp. Propionic

bacteria have also been shown to produce bacteriocins. Holo et al. (2002) identified

several bacteriocins produced from propionate producing bacteria.

Volatile fatty acids (VFAs) produced by bacteria have also been shown to display

bacteriostatic or bacteriocidal effects. Kunte et al. (1998) measured the effects of VFA

concentrations on the decimal reduction time (Tgo) of Salmonella typhi in anaerobic

digesters. The authors used a KVIC model floating dome digester (9.5 L) operated at a

15-day retention time and fed cattle dung slurry (6% total solids). The investigators

found that at a constant total VFA concentration (5000 mg/L) there was a rapid reduction

in S. typhi initially, but the numbers of the organism eventually leveled off and

maintained a concentration of 102 cells per ml. The authors reported a decrease in the

decimal reduction times between the control (T90, 4.22 days; VFA concentration, 100 -

125 mg/L) and experimental (T9o, 18.63 days; VFA concentration, 4800 5700 mg/L)

digesters. The study, however, did not determine the concentration of specific VFAs.

Kwon and Ricke (1999) specifically studied the effect of propionate on S. typhimurium as

a pure culture under anaerobic conditions. Details of the study are given previously (see

Chapter 2). Complete growth inhibition of S. typhimurium was observed at a propionate

concentration of 1830 mg/L. Sensitivity to VFAs under anaerobic conditions has also

been demonstrated with E. coli. Abdul and Lloyd (1985) showed that, under anaerobic









conditions, E. coli was inhibited by increasing concentrations (60 to 180 mM) of acetate,

propionate, and isobutyrate. The experimental conditions are described previously (see

Chapter 2).

Volatile fatty acid concentrations may contribute to indicator and pathogenic

organism decimation during anaerobic digestion. However, during anaerobic digestion,

VFAs, particularly acetate, serves as substrates for methanogenesis. Sooknah and Wilkie

(2004) found that the concentrations of acetate in the effluent of a fixed-film digester,

operated at a 2-day HRT and ambient temperature, treating flushed dairy manure

wastewater was below 170 mg/L or undetectable. Therefore, VFAs may not contribute to

indicator and pathogenic bacteria reduction during anaerobic digestion in a fixed-film

system.

The contribution of the indigenous microflora in the liquid phase during fixed-film

anaerobic digestion to indicator and pathogenic bacteria reduction may include the

production of antimicrobial compounds, more so than the production of VFAs. However,

competition between the indigenous microflora and indicator and pathogenic bacteria

may also occur. Thus, the role of indigenous microflora in the liquid phase during fixed-

film anaerobic digestion remains unclear.

Purpose

The purpose of the current experiment was to determine if the reduction of indicator

and pathogenic bacteria in the liquid phase of fixed-film anaerobic digestion at ambient

temperature is attributed to the presence of indigenous microflora, inhibition of growth

by digester contents, or by starvation.









Materials and Methods

Dairy Research Unit

The University of Florida Dairy Research Unit (DRU) is located in Hague, FL.

The facility has an average milking herd of 500 cows, which are housed and fed in free

stall barns. These barns are constructed with slanted concrete floors to allow downward

flow of the flush water to remove manure and urine. Sand is used as bedding in the free

stall barns instead of organic bedding materials because sand is inorganic and limits

bacterial growth.

Manure Handling

The manure handling process starts with flushing of the free stall barns and the

milking parlor. Manure is removed by intermittent, automated flushing. Sand is

removed by sedimentation in the sand trap and fibrous solids are removed using a

mechanical solids separator. The remaining solids are further removed by sedimentation

basins. The resulting slurry (influent) flows through a bar-screen before entering the

pump sump.

Fixed-film Anaerobic Digester

The fixed-film anaerobic digester used in the study is a demonstration-scale unit

with a 100,000 gal total capacity (97,377 gal active liquid volume). The system was

operated at ambient temperature and an average HRT of 3-days. The system is located

past the free stall barns and adjacent to the waste storage pond. The digester is equipped

with a flare and is used to convert excess methane to CO2 and H20, to avoid methane

emissions. Influent is fed into the digester from the pump sump. The material is recycled

through inert media to be degraded by the attached biofilm. Anaerobically digested

effluent is stored in waste storage ponds before being applied to land.









Sample Collection, Characterization, and Preparation

Sample Collection. Influent and effluent was collected from the DRU. Influent

was collected as three 1 L samples from the pump sump. Effluent was collected as three

1 L samples from a sampling port located on the side of the fixed-film reactor. The port

is used for taking samples of the effluent prior to exiting the system. Sample temperature

was measured at the time of collection.

Characterization. The samples were characterized by measuring the following

parameters according to standard methods (APHA, 1998): pH (section 4500-H B), total

solids (TS, section 2540 B), volatile solids (VS, section 2540 E), suspended solids (SS,

section 2540 D) and volatile suspended solids (VSS, section 2540 E). Total chemical

oxygen demand (TCOD) was performed on the whole fraction of samples using COD

tubes (Hach Co. Loveland, CO). Soluble chemical oxygen demand (SCOD) was

performed on the soluble fraction of wastewater. The soluble fraction was obtained by

centrifugation at 11,000 x g for 30 minutes and filtration through a 0.45 pm nitrocellulose

filter (Millipore). A 5-day biochemical oxygen demand (BOD5) was measured on the

whole (TBOD5) and soluble (SBOD5) fractions of the samples using a BODTrak

apparatus (Hach Co., Loveland, CO). To prevent nitrification, 175 mg 2-chloro-6

trichloromethyl pyridine (N-Serve # 253335, Hach Co., Loveland, CO) was added to

each BOD bottle.

Microbial Analysis. The microbial population (i.e., anaerobes, aerobes, and

facultative organisms) was determined. Total anaerobes and aerobes were measured by

direct plating onto plate count agar (Difco). Plating and incubation for total anaerobes

was performed in the anaerobic chamber (described later). All plates were incubated at









room temperature for 5 days. Total facultative organisms were determined by most

probable number (MPN) technique using fluid thioglycollate (Difco) tightly capped and

incubated as previously stated using aerobic conditions.

Organic acids and sugars were measured in the soluble fractions of influent and

effluent. The samples (100 ml) were ionized by adjusting to pH 11 with 5 N NaOH. The

samples were then concentrated using a microrotary film evaporator (EvapotecTm, #421-

4000, Labconoco, Lenexa, KS) at 100C. Concentrated samples were reconstituted with

deionized water, using 3 ml for influent and 2 ml for effluent. Reconstituted samples

were then filtered through a 0.45(m nitrocellulose filter (Millipore). The filtered samples

were analyzed using a Hewlett-Packard HPLC (HP 1090 Series II) equipped with a

refractive index (RI) detector and a UV monitor (210 nm).

Bacterial Cultures

Escherichia coli, Enterococcus spp., S. aureus, and Salmonella spp. used in the

study were isolated previously from influent. Escherichia coli 0157:H7 (ATCC 43888)

was obtained from the American Type Culture Collection. All cultures were grown in

3% (w/v) tryptic soy broth (TSB) overnight at 370C. The cells were harvested by

centrifugation at 2,000 x g for 10 minutes and washed three times with sterile IX

phosphate buffered saline, pH 7.5 (PBS). The cells were resuspended in 10 ml PBS and

stored at 50C until use.

Bacterial Quantification

Bacterial concentrations were determined by direct plate counts using mFC agar,

mE agar, mannitol salt agar (acriflavine added for whole fractions), and XLD agar for E.

coli (and fecal coliforms), Enterococcus spp., S. aureus, and Salmonella spp.,

respectively. All media was prepared according to the manufacturer's instructions.









Enterococcus spp. and S. aureus were incubated at 370C for 48 hours. Fecal coliforms

and E. coli were incubated at 450C overnight. Salmonella spp. was incubated at 370C for

24 hours. Total anaerobes and aerobes were quantified as described previously.

Anaerobic Conditions

Anaerobic conditions during incubation were maintained in an anaerobic chamber

(Coy, Grass Lake, MI), which was filled with a gas mixture of 80% N2 and 20% CO2.

Residual oxygen was removed from the chamber using hydrogen and palladium catalysts.

Pipettes and equipment were stored within the chamber to minimize oxygen

contamination. The chamber was also equipped with a forced air incubator.

Survival in Whole and Soluble Wastewater Fractions

The soluble fraction of influent was used to expose the test organisms to the

substrates present in the liquid phase during feeding of the digester. The soluble fraction

of effluent was used to expose the test organisms to the substrates that would be present

in the liquid phase during digestion. The soluble fractions of influent and effluent were

obtained as previously described in "Sample Collection, Characterization, and

Preparation." The soluble fractions were transferred as 5 ml aliquots in triplicate to

sterile, capped 13mm test tubes. The samples were inoculated individually with each test

organism, prepared as previously described. The samples were then transferred to the

anaerobic chamber and incubated for 3 days at 280C. Positive and negative controls were

3% TSB and PBS, respectively. Total anaerobes, aerobes, facultative organisms, and test

organisms were measured as previously described.

The whole fraction of influent was used to expose the test organisms to the

indigenous population that would be present in the liquid phase during feeding of the

digester. The whole fraction of effluent was used to expose the test organisms to the









indigenous population that would be present during digestion of the liquid phase. The

whole fractions of influent and effluent were transferred as 5 ml aliquots in triplicate into

sterile, capped 13mm test tubes and screw-cap tubes containing a magnetic stirring bar.

The tubes were transferred to the anaerobic chamber and incubated for 3 days at 28C

(without mixing) and 380C (mixed). Total anaerobes, aerobes, facultative organisms,

fecal coliforms, Enterococcus spp., and S. aureus were measured as previously described.

The survival of E. coli 0157:H7 and indigenous Salmonella spp. was not studied in

experiments with the whole fraction. Due to the low occurrence of these pathogens in the

wastestream of the DRU, artificial inoculation would have been required and potentially

would hinder simulating the natural population encountered by indigenous fecal

coliforms, Enterococcus spp. and S. aureus.

Inhibition and Wastewater Supplementation Studies

Inhibition effects were determined by supplementing the soluble fractions of

influent and effluent with 0.09% TSB. The amount of TSB used was the minimal

concentration that could support the growth of S. aureus. Soluble influent and effluent,

and supplemented soluble fractions were inoculated with a suspension of S. aureus

prepared as previously described. Negative and positive controls were PBS and 0.09%

TSB, respectively. The samples were transferred to the anaerobic chamber and incubated

under for 3 days at 280C. Staphylococcus aureus were quantified as previously

described.

Effluent was supplemented with a carbon source and yeast extract (YE) to

determine the effect of substrate limitation on the proliferation of S. aureus. Effluent was

supplemented with 5 and 55 mM glucose, 0.1% YE, and ashed YE. Glucose served as a

sole source of carbon and YE served to represent organic carbon and nutrients that may









be present in manure from cellular debris. The use of 55 mM glucose was based on the

carbohydrate concentration of commercial growth media (e.g., mannital salt agar, Difco).

The percentages of carbon in 5 mM and 55 mM glucose was equivalent to the percentage

of carbon in 0.1% YE (Table 3-1). Ashed YE served as a source of inorganic nutrients

and minerals. Ashed YE was obtained by ignition of 5g YE at 5500C for 4 hrs. The

residue following ignition was added to the effluent at the same amount present in 0.1%

YE. Soluble wastewater, supplemented soluble wastewater, and controls (i.e., PBS, 3%

TSB, and influent) were inoculated with S. aureus suspended in PBS. The samples were

transferred to the anaerobic chamber, incubated, and assayed for S. aureus as previously

described.

Statistical Analysis

Logio increases or decreases were determined under each condition by subtracting

the final loglo transformed concentration from the initial loglo transformed concentration.

Each condition was compared with the positive control and negative control using one-

way ANOVA and Duncan's Method with a significance level of 0.05. The statistical

analysis was performed using ProStat v 3.5.

Results

Wastewater Characteristics

The properties of the wastewater samples used in the study are given in Table 3-2.

Anaerobic treatment reduced TCOD by 52%, SCOD by 72%, TBOD5 by 62%, and

SBOD5 by 57%. Total aerobes, anaerobes, and facultative organisms were reduced by

62%, 73%, and 19%, respectively. Acetate and acetoin were reduced by greater than

80%.









Growth of Bacteria in Whole and Soluble Fractions of Wastewater

To determine if the indigenous microflora affected the concentrations of indicator

bacteria (fecal coliforms and Enterococcus spp.) and pathogenic bacteria (S. aureus,

Salmonella spp. and E. coli 0157:H7), influent and effluent with and without indigenous

microflora (i.e., whole and soluble fractions, respectively) were incubated under

anaerobic conditions for 3 days at 280C.

A. Total aerobic, anaerobic, and facultative bacteria

Soluble wastewater fraction. Growth of total aerobic, anaerobic, and facultative

bacteria in the soluble fractions of influent and effluent is shown in Table 3-3. Logio

increases in concentration of all groups were significantly lower (P < 0.05) in influent

than in 3% TSB. However there was no significant difference (P < 0.05) between the

logo increases in concentration between 3% TSB and effluent for all three groups.

Furthermore, there was more growth of all groups in soluble effluent than in soluble

influent. Although growth of all groups was lower in the influent, the logo increase for

both groups was significantly higher (P < 0.05) than the other organisms used in the

study. This trend is also observed with effluent.

Whole wastewater fraction. Loglo decreases in concentrations of indigenous total

anaerobic, aerobic, and facultative bacteria were observed in the whole fractions of

influent and effluent at 380C and 280C (Table 3-4). Unlike tests with the soluble fractions

where 10 to 20 pl of wastewater was used as an inoculum, the whole fractions were

incubated without any alterations. Indigenous concentrations of all groups may have

already been at a maximum in the whole fractions, where further incubation resulted in a

decline in population. The loglo reduction of total anaerobes between 38C and 280C in

influent was not significantly different (P < 0.05). This trend was also observed in the









effluent. The loglo reduction for total anaerobic bacteria was significantly lower (P <

0.05) in effluent at 380C and 280C than in influent at the same temperatures. Conversely,

the logo decrease for total aerobic bacteria was significantly lower (P < 0.05) in influent

at 380C and 280C and in effluent at 380C than in effluent at 280C. Also, the reduction of

total aerobes in influent at 380C and 280C was significantly lower (P < 0.05) than for total

anaerobes in influent at the same temperature. The reduction of total facultative

organisms at 380C in influent and effluent and 280C in influent was significantly higher

than at 280C in effluent. Furthermore, the reduction of total anaerobes and facultative

organisms in effluent at 280C was significantly lower (P < 0.05) than for total aerobes,

fecal coliforms, Enterococcus spp, and S. aureus (Table 3-5). These results suggest that

the anaerobic and facultative population is more stable and active in the effluent.

B. E. coli and fecal coliforms

Soluble wastewater fraction. Escherichia coli isolated from influent was chosen

as a representative of the fecal coliform population in influent and effluent. This

organism was able to proliferate in the soluble fractions of influent and effluent (Table 3-

3). However, growth in both fractions was significantly lower (P < 0.05) than in the

positive control. Furthermore, growth of this strain of E. coli in effluent was significantly

lower (P < 0.05) than in influent. This result shows that effluent is limited in the amount

of available substrate to support significant increases in concentration.

Whole wastewater fraction. In the whole fractions of influent and effluent, fecal

coliforms were not detected after incubation at 380C or 28C (Table 3-4). This result

shows that in presence of indigenous total aerobic and anaerobic organisms, fecal

coliforms were not able to survive in influent or effluent at either temperature.









C. Enterococcus spp.

Soluble wastewater fraction. Enterococcus spp. isolated from influent was able to

proliferate in both soluble fractions of influent and effluent (Table 3-3). There was no

significant difference (P < 0.05) between the loglo increase in influent and the positive

control. However, the logo increase in effluent was significantly lower (P < 0.05) than

influent and the positive control. These results show that anaerobic treatment reduces the

amount of available substrate to support significant increases in concentration of

indigenous Enterococcus spp.

Whole wastewater fraction. There was significantly more reduction of

Enterococcus spp. in influent at 380C than at 280C (Table 3-4). Also, there was

significantly more reduction in influent at both temperatures than in effluent. The

reduction of Enterococcus spp. in effluent at 380C and 280C was not significantly

different (P < 0.05) (Table 3-4). There were significantly more reduction of

Enterococcus spp. in influent at both temperatures than for total aerobes and anaerobes

(Table 3-5). In the effluent at both temperatures, the reduction of Enterococcus spp. was

significantly higher (P < 0.05) than for total anaerobes (Table 3-5). In the whole

fractions of influent and effluent, the loglo reductions of Enterococcus spp. were 1.04 and

0.69, respectively (Table 3-4). This result shows that the indigenous microflora in

influent and effluent contributes to the reduction of this organism during the digestion

process. These results also suggest that mesophilic temperatures are not required to

reduce Enterococcus spp.









D. Salmonella spp.

Soluble wastewater fraction. Salmonella spp. isolated from influent was able to

proliferate in the soluble fractions of influent and effluent (Table 3-3). However, growth

in both fractions was significantly lower (P < 0.05) than the positive control.

E. E. coli 0157:H7

Soluble wastewater fraction. Escherichia coli 0157:H7 was able to proliferate in

the soluble fractions of influent and effluent (Table 3-3). Also, there was no significant

difference (P < 0.05) between the logo increases in concentrations for the positive

control, influent and effluent.

Differences in growth were observed between pathogenic and nonpathogenic E.coli

in the soluble fractions of influent and effluent. Growth in influent was similar to that of

indigenous E. coli. However, the logo increase in effluent for E. coli isolated from

influent was significantly lower (P < 0.05) than for E. coli 0157:H7.

F. S. aureus

Soluble wastewater fraction. There was no significant difference (P < 0.05) in

the growth of indigenous S. aureus in the soluble fraction of influent and the positive

control (Table 3-3). There was also no significant difference (P < 0.05) between the logio

decreases in effluent and the negative control. This shows that S. aureus can proliferate

in influent but not effluent.

Whole wastewater fraction. Reductions of S. aureus in influent and effluent at

380C and 280C were similar (Table 3-4). Also, the reduction of S. aureus in effluent at

380C and 280C was not significantly different (P < 0.05). These results suggest that

digestion at both temperatures result in similar decreases of S. aureus in effluent.









However, there was significantly more reduction of S. aureus in influent at 380C than at

280C.

The reduction of S. aureus in effluent at 380C and 280C was significantly higher (P

< 0.05) than for total anaerobes and aerobes (Table 3-5). However, the reductions of S.

aureus observed in the whole fraction of effluent at both temperatures (-0.84) were less

than those observed in the soluble fraction (-1.43) at 280C. These results suggest that

either inhibitory compounds or nutritional limitations adversely impact S. aureus.

Inhibition or starvation of S. aureus. To determine if inhibitory compounds or

substrate limitations impacted S. aureus, soluble effluent was supplemented with a

minimal concentration of TSB. As previously observed, S. aureus was not able to

proliferate in effluent (Table 3-6). However, supplementation with TSB resulted in a 1.6

loglo increase in concentration. This result suggests that proliferation of S. aureus is

restricted by substrate limitations, not inhibitory compounds.

Wastewater supplementation. Supplementation resulted in proliferation of S.

aureus in soluble effluent (Table 3-7). Log1o increases of S. aureus in effluent

supplemented with 55 mM glucose solution were significantly higher (P < 0.05) than in

the other solutions tested. Growth was not observed in effluent or 5mM and 55 mM

glucose solutions. However, addition of 5 mM and 55 mM glucose to effluent resulted in

proliferation of S. aureus. Also, there was no growth detected in effluent supplemented

with ashed yeast extract, suggesting that inorganic nutrition is not the limiting substrate.

These results demonstrate that the inability of S. aureus to proliferate in the effluent is

due to the lack of sufficient sources of carbon.









Discussion and Conclusions

Anaerobic digestion is known to reduce the concentrations of pathogenic bacteria

from various manures. The decimation is commonly attributed to operating temperature

and HRT, where higher temperatures and longer HRTs result in higher rates of

decimation. However, manure contains a diverse microbial population and the influence

of the indigenous microflora in manure on the survival of indicator organisms and

pathogenic bacteria has not been studied extensively. Few studies have investigated the

influence of indigenous microflora on pathogenic bacteria in manure, under anaerobic

conditions. Shin et al. (2002) found that a fecal suspension (FS) and its anaerobic culture

(FC) displayed inhibitory effects on E. coli 0157:H7. An overnight culture ofE. coli

0157:H7 (1 x 105 CFU/ml) was grown in mixed culture with FS (5 x 108 CFU/ml) and

FC (5 x 108 CFU/ml) anaerobically for 24 h at 370C. Growth of these pathogenic bacteria

was inhibited in the presence of FS and FC. Also, decimation of E. coli and Salmonella

spp. during mesophilic anaerobic digestion of sewage sludge (20 day HRT) has been

attributed to competition with indigenous microflora (Smith et al., 2005).

Similar impacts ofindigeous microflora have been demonstrated against S. aureus.

Donnelly et al. (1968) studied the proliferation and enterotoxin production of S. aureus

(104 CFU/ml) under aerobic conditions in raw milk with a high (> 106 CFU/ml) and low

(< 104 CFU/ml) standard plate count (SPC, i.e., indigenous microflora). At 300C, S.

aureus was able to proliferate and produce toxin in low SPC milk. However, in high SPC

milk at the same temperature, S. aureus demonstrated less proliferation and no

enterotoxin was detected. The authors concluded that S. aureus was adversely impacted

by the activity of indigenous microflora. In the current study, the indigenous microflora

in wastewater (107 CFU/ml) caused decimation of native S. aureus (103 CFU/ml).









DiGiacinto and Frazier (1966) demonstrated inhibited growth of S. aureus during

incubation with coliforms and Proteus spp. under aerobic conditions in TSB.

Eschericihia coli (2 x 104 CFU/ml) and Proteus vulgaris (2 x 104 CFU/ml), both

commonly found in animal manure, were found to inhibit the growth of S. aureus (2 x

104 CFU/ml) in mixed cultures at 22C and 300C. Results from the current study showed

significant reduction of fecal coliforms (> 2 logo) in the presence of indigenous

microflora, suggesting their competition with S. aureus may be minimal. However,

competition between S. aureus and Proteus spp. during fixed-film anaerobic digestion is

not known.

The current study suggests that the indigenous microflora responsible for

decimation of indicator and pathogenic bacteria may consist of strict anaerobic and

facultative organisms. Total anaerobic and facultative organisms were reduced

significantly less than fecal coliforms, Enterococcus spp., and S. aureus. These results

suggest that the activity of the anaerobic and facultative population in the liquid phase

contributes to the reduction of indicator and pathogenic bacteria during fixed-film

anaerobic digestion.

In the current study, the test organisms, with the exception of S. aureus, were able

to proliferate in the soluble fraction of effluent, whereas growth did not occur in the

whole fraction of effluent. The difference in growth may be attributed to the ability of

the test organisms, as a pure culture, to use the remaining carbon sources (e.g., acetate

and acetoin). The concentration of the remaining acetate (1.1 mM) and acetoin (0.4 mM)

may have been too low to support the proliferation S. aureus.









Exposure to optimum growth temperature of bacterial pathogens, in the presence of

indigenous microflora, does not increase the proliferation of pathogenic bacteria

(Donnelly et al., 1968). Similarily, in the current study, the reductions of indicator and

pathogenic bacteria observed in the whole fraction of effluent at 380C and 280C were

similar. These results suggest that anaerobic digestion at optimum growth temperatures

of indicator and pathogenic bacteria will not increase survival in the presence of

indigenous microflora.

Limited substrate concentration was found to specifically affect S. aureus. In the

current study, S. aureus was the only test organism that could not proliferate in the

soluble fraction of effluent, where growth was observed in the soluble fraction of

influent. Furthermore, a higher reduction was observed in the soluble fraction of effluent

than in the whole fraction of effluent, suggesting that removal of nutrients or the presence

of inhibitory compounds, resulting from the activity of indigenous microflora, influenced

reduction of S. aureus during incubation. Supplementation with tryptic soy broth

demonstrated that starvation, and not inhibitory compounds, were preventing

proliferation of S. aureus. This finding suggests that substrates necessary to support S.

aureus were removed during anaerobic treatment of influent, resulting in starvation of S.

aureus. Furthermore, because S. aureus was the only test organism that did not grow in

the soluble fraction of effluent, the use of the tested indicators to predict the survivability

of S. aureus following digestion may not be valid. Therefore, direct monitoring of S.

aureus, as apposed to using surrogates, may be necessary.

Starvation of S. aureus during anaerobic digestion may be induced by the inability

to compete with the indigenous microflora. The inability to compete with the indigenous






46

microflora may be attributed to the differences in concentrations in native wastewater.

The indigenous population was present at 106 to 107 CFU/ml and S. aureus was present at

102 to 103 CFU/ml. Furthermore, during digestion, there was a low reduction (19%) of

facultative organisms, suggesting their stability in the liquid phase during digestion.

Thus, S. aureus may have been out competed for available substrates, resulting in

starvation.

Although S. aureus may be reduced by starvation, survival of the residual

population during digestion may be possible. In stressed conditions, S. aureus enters a

starvation-survival state. The starvation state of S. aureus was characterized by Clements

and Forster (1998). Differential protein expression and morphological changes of S.

aureus were observed after the cells were exposed to long-term glucose limiting

conditions. The morphological changes associated with the starvation state included

reduction in cell size and lack of division septa. Several different proteins not normally

observed in vegetative cells were expressed prior to and during the starvation state. This

finding suggested that S. aureus utilized differential protein expression to enter the

starvation state and that the cells were not dormant during the starvation state. Also,

during starvation conditions, a 3 logo reduction of vegetative S. aureus was observed

before entry into the starvation state. The investigators found that the recovery of S.

aureus from a starvation state could not occur by the addition of solely glucose or amino

acids. A combination of glucose and amino acids were required to induce growth. Also,

an unknown factor (or factors) present in media utilized by vegetative S. aureus cells was

found to induce growth of starved cells.









Another phenotypic trait of starvation-state S. aureus is the formation of smaller

colonies, as compared to normal vegetative cells, that are similar to small colony variants

(Clements et al., 1999). Small colony variants of S. aureus are mutations that arise from

stressed conditions. The mutations result in decreased coagulase and hemolysin activity,

slow growth, reduced or no pigmentation, and small colony size (diameter < 0.1 mm) on

agar media (Clements and Foster, 1999, Kaplan and Dye, 1976).

In the current study, the results demonstrated proliferation of S. aureus in the

soluble fraction of effluent following the addition of only carbon. This result shows that

S. aureus was starved for carbon and that carbon is a growth-limiting constituent in

effluent. During anaerobic digestion, carbon is converted mainly to CH4, CO2, and

biomass. The reduced COD of the effluent demonstrated the reduction of carbon during

anaerobic digestion. The reduced COD suggests that utilization of carbon by other

indigenous microflora during anaerobic digestion resulted in starvation of S. aureus.

Entry into a starvation stated during anaerobic digestion may occur when the organism

becomes stressed.

The current study shows the reduction of indicator and pathogenic bacteria in the

liquid fraction is due to microbial competition and starvation from the lack of sufficient

carbon sources. These findings suggest possible mechanisms for the decimation of these

organisms during anaerobic treatment. The current study showed reductions of these

organisms in the presence of indigenous microflora at ambient temperature (280C) and a

low HRT (3 days). The results of this study demonstrate the importance of indigenous

microflora in manure for sanitization of manure wastewater by anaerobic digestion.

Furthermore, removal of carbon sources necessary to support pathogenic bacteria may






48


play a role in decimation. Also, the use of indicators to predict the presence ofS. aureus

should be reevaluated.










Table 3-1. Percentage of carbon from various sources in yeast extract.


Component* # Carbons Mol. Wt. % Carbon


Ala
Arg
Cys
Asp
Glu
Gly
His
He
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
carbohydrates
Biotin
Choline chloride
folic acid
Inositol
nicotinic acid
PABA
Pantothenic acid
Pyridoxine
Riboflavine
Thiamine
thymidine


89
174
121
133
147
75
155
131
131
146
149
165
115
105
119
204
181
117
180
244.31
139.63
441.4
180.16
123.11
137.00
219.23
205.64
376.36
300.81
242.23


40.48
41.41
29.78
36.12
40.85
32.03
46.49
55.01
55.01
49.36
40.30
65.51
52.22
34.31
40.37
64.76
59.72
51.32
40.03
46.19
43.01
51.70
40.00
58.53
47.98
49.30
46.72
54.25
42.73
49.58


% Carbon from
% component in componfrom
YE component in
YE
5.36 2.17
3.02 1.25
0.74 0.22
6.69 2.42
14.20 5.80
3.25 1.04
1.20 0.56
3.23 1.78
4.69 2.58
5.15 2.54
1.05 0.42
2.53 1.66
2.60 1.36
2.84 0.97
2.95 1.19
1.36 0.88
1.20 0.72
3.79 1.95
17.50 7.01
0.0003 0.00015
0.03 0.013
0.0002 0.00008
0.14 0.056
0.060 0.035
0.076 0.037
0.027 0.014
0.004 0.002
0.012 0.006
0.053 0.023
0.002 0.0009


TOTAL % C in YE
Amount (%C) in 1% YE
Amount (%C) in 55 mM carbon source
Amount (%C) in 0.1% YE
Amount (%C) in 5 mM carbon source


36.69
0.367
0.390
0.037
0.036


*The components of yeast extract (YE) were obtained from a typical analysis of YE (Difco Laboratories,
1998).










Table 3-2. Characterization of samples used for all experiments.


Parameter*
Temperature, C

pH

TCOD, mg/L

SCOD, mg/L

TS, mg/L

SS, mg/L

VS, mg/L

VSS, mg/L

TBOD5, mg/L

SBODs, mg/L


Total aerobes, CFU/ml

Total anaerobes, CFU/ml

Total facultative, MPN/ml


Acetate, mM

Acetoin, mM


2.1 + 0.2x107

1.3 + 0.4 x 107

9.4+0.3x 106


6.3 + 0.7

2.1 +0.6


7.9+ 1.0 x 10

3.4 + 1.1 x 106

7.6+0.5x 106

1.1+0.07

0.42 + 0.02


Notes: Anaerobically digested flushed dairy manure wastewater (effluent) was obtained from the
University of Florida IFAS fixed-film anaerobic digester. Flushed dairy manure wastewater (influent) was
material collected prior to anaerobic treatment.

*Parameter abbreviations are as follows: TCOD = total chemical oxygen demand, SCOD = soluble
chemical oxygen demand, TS = total solids, SS = suspended solids, VS = volatile solids, VSS = volatile
suspended solids, TBOD5 = 5-day total biochemical oxygen demand and SBOD5 = 5-day soluble
biochemical oxygen demand.


**Values are average standard deviation (n = 3).


Influent


3707 + 15**

2083 + 38

3904 + 169

1136 + 69

2352 + 114

920 +22

3413 + 175


1046+5


Effluent
25

7.06

1779 + 31

590 + 4

3064 + 55

1034 + 69

1543 +40

780 + 65

1313+64

448+33










Table 3-3. Growth of indicator and pathogenic bacteria in the soluble fraction of
wastewater.




organism Initial conc, Loglo Reduction
Organism CFU/ml
3% TSB IX PBS Influent Effluent

Total aerobes 3.6 + 0.6 x 104 4.10A NA 2.60B 3.99A

Total anaerobes 1.7 + 0.2 x 104 3.54^ NA 2.86B 3.63A


Total facultative, 8.3 + 0.3 x 103 3.98A NA 2.95c 4.15A
MPN/ml


E. coli 3.9 + 0.6 x 106 1.48A -0.54D 0.74B 0.34c


Enterococcus 6.3 + 0.2 x 106 0.54A < -5.00 0.41A 0.268
spp.


S. aureus 7.9 + 0.2 x 105 0.33A -1.43B 0.50A -1.43B


Salmonella spp. 2.5+0.1 x 106 2.00A < -5.00 0.91c 1.11"


E. coli 0157:H7 6.4 + 0.3 x 106 0.80A -0.71B 0.66A 0.67A


*Notes: The soluble fraction of influent and effluent were collected as described in the text. A small
volume (20 pl) of influent was used as the inoculum for total aerobic, anaerobic, and facultative organisms.
The filtrate was inoculated with each test organism and incubated without mixing under anaerobic
conditions for 3 days at 28C. Concentration values are average + standard deviation (n=3).

NA, not applicable.

Values with the same letter within the same row are not significantly different (P < 0.05).









Table 3-4. Growth of indigenous indicator bacteria and S. aureus in the whole fraction of
wastewater.



Logo Reduction
Influent Effluent
Organism
Initial conc, 28 Initial conc, 380C 280C
CFU/ml 3C 2C CFU/ml 38C 28C


Total anaerobes 1.3 + 0.4 x 107 -0.5B,c -0.6c 3.4 + 1.1 x 106 -0.2A -0.4AB


Total aerobes 2.1 + 0.2 x 107 -0.2A -0.1A 7.9+ 1.0 x 106 -0.1A -6B


Total facultative 9.4 + 0.3 x 106 -1.0B -1.4c 7.6 + 0.5 x 106 -1.0B -0.4A


Fecal coliforms 2.4 + 0.3 x 105 < -4.0 < -3.0 2.8 + 0.2 x 104 < -3.0 < -2.0


Enterococcus 7.2 + 1.2 x 10' -1.3c -1.0B 2.2 + 1.2 x 10 -0.8A -07A
spp. _


S. aureus 1.3 + 0.6 x 103 -0.9B -0.7A 1.0 + 0.1 x 102 -0.8AB -0.8AB



A-C Values with the same letter in the same row are not significant (P < 0.05)

Notes: Influent and effluent samples were incubated with under anaerobic conditions for 3 days at 380C
(mixing) or 280C (static). Total anaerobes, aerobes, fecal coliforms, Enterococcus spp., and S. aureus were
measured as described in the text. Concentration values are average + standard deviation (n=3).











Table 3-5. Comparison of growth between indigenous bacteria in wastewater at 38C and
280C.



Loglo Reduction
Organism Influent Effluent

380C 280C 380C 280C


Total anaerobes


Total aerobes


Total facultative


Fecal coliforms


Enterococcus spp.


S. aureus


-0.5


-0.2A


-1.0c


< -4.0


-1.3D


-0.9c


-0.


-0.


-1.


< -


-1.


-0.


6 -0.2^A


1A A0.1A


4D -1.0B


3.0 < -3.0


0c -0.8"


7B -0.8'B


-0.4A


-0.6B


-0.4A


< -2.0


-0.7'c


-0.8c


A-D Values with the same letter in the same column are not significant (P < 0.05)

Notes: Influent and effluent samples were incubated under anaerobic conditions for 3 days at 38C
(mixing) or 28*C (static). Total anaerobes, aerobes, fecal coliforms, Enterococcus spp., and S. aureus were
measured as described in the text.









Table 3-6. Determination of inhibitory or nutritional limitation on the growth of
indigenous S. aureus.



Initial conc, Final conc, Logo0 Logio contribution of
ConditionCFU/ml CFU/ml increase or .09% TSB
(decrease)

0% TSB 3.4 + 0.3 x 104 1.3 + 0.1 x 103 -1.4

0.09 % TSB 6.1 + 0.5 x 103 1.0 + 0.2 x 07 3.2^

Influent 2.1 + 0.4 x 104 4.4 + 0.4 x 105 1.3


Influent + 1.7 + 0.2 x 104 4.3 + 0.7 x 10 2.4B 1.1B
0.09% TSB
Effluent 1.5 + 0.3 x 104 2.5 + 0.2 x 103 -0.8E

Effluent + 1.6 + 0.4 x 104 6.4 + 1.6 x 105 1.6c 2.4
0.09% TSB



Notes: The soluble fraction of influent and effluent were collected by centrifugation at 11,000 x g for 30
mins. The supernatant was filtered using a 0.45 numm nitrocellulose filter. The filtrates were supplemented
with tryptic soy broth (TSB) to a final concentration of 0.09%. The filtrates, supplemented filtrates, and IX
PBS (0% TSB) were inoculated with S. aureus isolated from influent and incubated without mixing under
anaerobic conditions for 3 days at 28C. Samples were assayed daily for test organisms. Concentration
values are average + standard deviation (n=3).
A- F Values with the same letter within the same column are not significantly different (P < 0.05).







55


Table 3-7. Growth of S. aureus in soluble effluent and soluble effluent with various
amendments.


Condition Initial conc, CFU/ml Final conc, CFU/ml Log increase or
decrease
1X PBS 1.1 + 0.15 x 104 5.2 + 1.2 x 10' -0.5

3% TSB 9.7 + 1.5 x 103 5.4 + 1.0 x l07 3.3B

Influent 1.2 + 0.25 x 104 3.1 + 0.38 x l07 2.4D

Effluent 7.0 + 2.0 x 103 2.8 + 0.3 x 103 -0.4

Glucose, 5 mM 9.7 + 1.2 x 10' 4.8 + 3.2 x 102 -1.4

Effluent + glucose, 5 mM 9.7 + 1.2 x 103 7.7 + 1.5 x 104 0.9E
Glucose, 55 mM 6.0 + 1.3 x 103 2.2 + 0.53 x 103 -0.4

Effluent + glucose, 55 mM 1.6 + 0.31 x 104 9.5 + 0.33 x 107 3.6A
Yeast Extract, 0.1%* 2.6 + 0.36 x 104 2.3 + 0.93 x 107 2.5c
Effluent + Yeast Extract, 0.1% 2.9 + 0.15 x 104 2.4 + 0.66 x 107 2.9c
Ashed Yeast Extract 3.2 + 0.7 x 104 1.6 + 0.2 x 104 -0.3G


*Yeast extract at 0.1% is equivalent to 5 mM glucose on a carbon basis (see Table 3-1).

Notes: Phosphate buffered saline (IX PBS), 3% tryptic soy broth (TSB), influent, effluent, supplemented
effluent, and supplement solutions in sterile saline were inoculated with S. aureus suspended in PBS.
Samples were incubated without mixing under anaerobic conditions for 3 days at 28C. Concentration
values are average + standard deviation (n=3).
A -H Values with the same letters within the same column are not significantly different (P < 0.05).














CHAPTER 4
INDICATOR AND PATHOGENIC BACTERIA REDUCTION DURING
ANAEROBIC DIGESTION: ATTACHMENT TO THE FIXED FILM

Introduction

Fixed-bed anaerobic digesters are systems that use internal support media where

anaerobic bacteria attach to the surface and proliferate, resulting in the formation of a

biofilm (see Chapter 2). The retained biofilm (i.e. fixed-film) allows for a higher

treatment capacity as compared to systems that do not use internal support media

(Ramasamy and Abbasi, 2000). The increased treatment capacity for systems with

internal support media is due to the retention of methanogens, which grow at a slow rate

(Wilkie and Colleran, 1989). Digesters that are not constructed with an internal support

media can potentially lose a substantial portion of the methanogenic population as

effluent exits the system. To maintain treatment efficiency, such systems have to operate

at long retention times (> 10 days) to reestablish a methanogenic population.

Pathogen reduction by anaerobic digestion has been well studied in various

systems, including a fixed-film system (Barnes, 2002, Davis et al., 2001, Hill, 2003).

However, for fixed-film systems, the role of the retained biofilm in pathogen reduction

during anaerobic digestion remains unclear.

Purpose

The purpose of the current study was to use a model organism to determine if

indicator and pathogenic bacteria were removed from the liquid phase during anaerobic

digestion by attachment to the fixed-film.









Materials and Methods

Pilot-Scale Fixed-Film Reactors

The two pilot-scale fixed-film reactors used for biofilm attachment studies had a

total capacity of 104 gal each. Both reactors were operated at ambient temperature (28C

to 330C) and a 3-day HRT. The systems were considered at steady state once SCOD

reduction was consistent (69% + 5%). Both reactors were packed with vertically

orientated PVC pipes to provide internal support media for biofilm formation. Each

system was continuously fed influent from a feeding manifold by a peristaltic pump

(MasterFlex). However, one unit was operated in up-flow mode, where contents

entered the reactor from the bottom and the other was operated in down-flow mode,

where contents entered the reactor from the top. Contents of both reactors were mixed,

by recycling at a 3:1 ratio to feed rate, by another peristaltic pump.

Bacterial Culture

Escherichia coli BL(21)DE3 haboring plasmid plAM1055, for the production of

green fluorescent protein (GFP), was grown at 37C overnight in LB broth, Lennox

(Fisher) with 0.4 mM isopropyl 3f-D-l-thiogalactopyranoside (IPTG) (Amresco, Solon,

OH) and 15.4 pg/ml of ampicillin (Sigma). The cells were harvested by centrifugation at

2,000 x g for 10 minutes and washed three times with sterile IX PBS. The cells were

resuspended in 100 ml IX PBS and stored at 5C until use.

Survival Comparability with Indigenous Fecal Coliforms

Influent and effluent samples were collected as previously described (see Chapter

3). GFP producing E. coli were inoculated into whole fractions of influent and effluent.

Samples were incubated under anaerobic conditions (see Chapter 3) for 3 days at 280C.

Indigenous fecal coliforms were assayed daily as previously described (see Chapter 3).









GFP producing E. coli were assayed daily using plate count agar supplemented with

0.00002% crystal violet (Carolina Biological Supply Co., Burlington, NC), 0.4 mM

IPTG, and 15.4 pg/ml ampicillin. Following incubation, GFP producing E. coli colonies

were enumerated by exposing the plates to a UV light source (366 nm).

Biofilm Attachment Studies

A 100 ml suspension of GFP producing E. coli (1.2 x 107 CFU/ml) was prepared as

previously described. The suspension of bacteria was fed into the reactor through the

feeding pumps. Effluent samples from the reactor were collected at 1 h intervals over 12

h, and then a composite sample was taken daily. Serial dilutions of effluent samples were

done using IX PBS. Viable counts of GFP producing E. coli were performed by direct

plating diluted samples onto plate count agar prepared and incubated as previously

described. Total counts of GFP producing E. coli were performed by filtering diluted

samples through a 0.45 gm polycarbonate filter (47 mm, Nucleopore Corp., Pleasanton,

CA). Filters were mounted on glass slides (50 mm x 75 mm) and cells were enumerated

under 250X total magnification with a fluorescent microscope (Carl Zeiss, Standard 25)

equipped with a UV light source (470 nm). The total number of test organisms expected

to be present in the effluent at each sampling event was calculated by the following:

Predicted effluent volume = (reactor volume / HRT) x sampling time interval

Predicted effluent CFU = (initial CFU / reactor volume) x predicted effluent volume

Biofilm Sampling and Examining

Support media in close proximity to where influent entered each system were

chosen for sampling. Biofilm samples were obtained by removing selected support

media from each reactor. Then, 2 in sections were cut from the bottom (up-flow reactor)

and top (down-flow reactor) of the support media. Each 2 in section was then divided into









smaller sections and placed inside containers lined with moist paper towels to prevent

desiccation. The sections were examined under 300X total magnification with a

stereomicroscope (Leica MZ75) equipped with a UV light source (470 nm). Photos of the

samples were taken using a digital camera (Nikon Coolpix 4500) connected to the

microscope by a lens attachment (Martin Microscope mmcool, S/N: 0941).

Results

Fecal Coliform and GFP E. coli Survival

The GFP producing E. coli displayed comparable survival with indigenous fecal

coliforms in both the influent and effluent, suggesting that the test organism could serve

as a suitable model for indigenous fecal coliforms (Figure 4-1).

Biofilm Attachment Studies

Each pilot-scale fixed-film reactor was fed a suspension of GFP producing E. coli.

The number of cells detected at each sampling event in the up-flow reactor was lower

than the predicted values (Figure 4-2). There was a 1 logo difference between the

predicted values and the experimental values at each sampling event. Also, the model

organism could not be detected on Day 4 and after Day 6. After 1 HRT (3 days), there

was a 0.9 loglo reduction of the model organism.

During the first 12 h, retention of the model organism in the down-flow reactor was

similar to the upflow-reactor (Figure 4-3). However, on Day 1, 4 and 6, the experimental

values were similar to the predicted values. The model organism was not detected after

Day 7. After 1 HRT, there was a 0.2 logo reduction of the model organism.

To confirm the attachment of the model organism to the fixed-film, biofilm

samples were obtained and examined. GFP producing E. coli were detected on the fixed-

film (Figure 4-4). However, the viability of the attached organisms was not determined.









Discussion and Conclusions

The current study demonstrates the role of the fixed-film for reducing indicator and

pathogenic bacteria during digestion. Our results show that the test organism attached to

the fixed-film, thereby reducing effluent concentrations of the test organism. However, a

difference in retention was observed between the two modes of operation. After 3 days

of operation, the reduction in the up-flow mode (0.7 loglo) was higher than in the down-

flow mode (0.1 logoo. Conversely, Barnes (2002) reported a 0.2 logo reduction of fecal

coliforms, using the same reactor operated in up-flow mode at the same conditions as the

current study. Also, in the study, a similar reduction (0.4 logo) of fecal coliforms was

achieved during operation in down-flow mode. However, in the current study, the

difference between the two modes suggests that operation in up-flow mode may lead to

more interaction between the fixed-film and indicator and pathogenic organisms.

Anaerobic systems that employ fixed-beds have demonstrated higher treatment

efficiencies than systems without fixed-beds. However, the contribution of the fixed-film

to pathogen reduction from various substrates has not been extensively studied. The

current study provided evidence that the fixed-film contributes to the reduction of

indicator and pathogenic bacteria during anaerobic digestion. After 3 days of operation, a

0.7 logo reduction was achieved. Previously, microbial competition was shown to

account for more than 2 logo reduction of indicator bacteria in batch studies (see Chapter

3). However, the attachment studies were performed with a continuously fed system,

where substrates and organisms were constantly introduced. In continuously fed systems,

microbial competition may still have a significant contribution to indicator and pathogen

reduction, but the constant influx of substrates and organisms may decrease this effect, in

comparison to the batch studies. However, in the current study, model organisms









detected in the effluent were not viable. This result suggests that inactivation of the

model organism occurred in the liquid phase during digestion. Previous results (see

Chapter 3) suggest that the inactivation in the liquid phase observed in the current study

may be due to microbial competition. In a continuously fed system, the fixed-film

provides a physical means for removal, whereas microbial competition may inactivate

unattached organisms in the liquid phase. The combination of attachment to the fixed-

film and microbial competition may synergistically contribute to the reduction of

indicator and pathogenic bacteria during digestion in the fixed-film reactor.








62





1.2-


1 -


0.8 --FC A
-0-GFP
o
0.6 -


0.4-


0.2 -


0
1.2 -


I -


0.8 -- FC

----GFP B
0.6


0.4


0.2 -


0
0 1 2 3

Time, day





Figure 4-1. Survival of indigenous fecal coliforms and GFP producing E. coli in
wastewater.

Notes: Influent (A) and effluent (B) were inoculated with GFP producing E. coli suspended in PBS.
Samples were incubated under anaerobic conditions for 3 days at 280C. Fecal coliforms (0) and GFP
producing E. coli ([) were assayed daily as described in the text. Each point is an average and error bars
represent standard deviation (n=3).












1.0E+09 -
S.OE+08 -
1.0E+07
1.0E+06 -
1.0E+05 -
1.0E+04-
1.0E+03 Predicted concentration
S.0E+02 -D- Experimental concentration
1.0E+01 -A-Predicted 1 logl 0 reduction
1.OE+00 I.. I I I 1..
0.08 0.13 0.17 0.21 0.38 0.42 0.46 0.5 1 3 4 6 7 8 9
Time, days


Figure 4-2. Retention of GFP E. coli within the pilot-scale fixed-film anaerobic digester
operated in up-flow mode.

Notes: A pilot-scale fixed-film reactor was fed a suspension of GFP producing E. coli. Effluent samples
were collected and total counts of GFP producing E. coli were performed as described in the text. Each
point represents triplicate samples and error bars represent standard deviation.











1.OE+09 -

1.0OE+08 -

1.0E+07 -

1.0E+06 -

1.0E+05 -

1.OE+04 -

1.0OE+03 --- Predicted concentration

1.0E+02 -0- Experimental concentration

1.OE+01 -A- Predicted 1 logl0 reduction

1.0E+00 -. I III.. .
0.08 0.13 0.17 0.21 0.38 0.42 0.46 0.5 1 3 4 6 7 8 9
Time, days



Figure 4-3. Retention of GFP producing E. coli within the pilot-scale fixed-film operated
in down-flow mode.

Notes: A pilot-scale fixed-film reactor was fed a suspension of GFP producing E. coli. Effluent samples
were collected and total counts of GFP producing E. coli were performed as described in the text. Each
point represents triplicate samples and error bars represent standard deviation.

















































Figure 4-4. GFP producing E. coli attached to the surface of the biofilm.

Notes: Samples of internal support media were removed from the pilot-scale fixed-film reactor. Samples
were cut from support media and viewed under 300 X total magnification with a UV light source (470 nm).
White arrows indicate GFP producing E. coli.














CHAPTER 5
BACTERIOPHAGE REDUCTION DURING ANAEROBIC DIGESTION: THE ROLE
OF INDIGENOUS MICROFLORA

Introduction

Animal manure may contain pathogenic viruses that pose severe human and herd

health concerns (Bicudo and Goyal, 2003, Pell, 1996). Therefore, treatment of manure is

necessary to reduce the levels of these organisms. Anaerobic digestion can effectively

reduce the concentration of viruses from various substrates, including manure. Berg and

Berman (1980) found that mesophilic and thermophilic anaerobic digestion (20 day

HRT) of domestic sludges achieved a 1 logo and 3 logo0 reduction, respectively, of

viruses after 20 days. Similarly, Huyard et al. (2000) investigated virus reduction in

sludge by a two-phase anaerobic digester (TPAD) operated at thermophilic (2 day HRT)

and mesophilic (10 day HRT) temperature. The authors reported a 4 loglo reduction of

poliovirus after 10 days. Also, Aitken et al. (2005) demonstrated that thermophilic

anaerobic digestion, at a 4 day HRT and treating sludge, achieved > 4 logo reduction of

poliovirus. However, reduction of male-specific bacteriophages was 1 to 2.6 logo.

Spillman et al. (1987) reported inactivation rates of less than 1 logo day"' for viruses

during mesophilic anaerobic digestion of sludge. Inactivation rates during thermophilic

digestion varied between 0.2 to > 8 logo hr-'. However, the authors did not report the

HRT of the digester used in the study.

Virus reductions similar to the aforementioned studies have been found during

treatment of animal manure. Lund et al. (1996) found that mesophilic anaerobic









digestion (15 day HRT) of mixed animal manure (25% pig, 75% cow) achieved a 4 logo

reduction of bovine enterovirus and porcine parvovirus after 1 and 2 days, respectively.

Derbyshire et al. (1986) examined the occurrence of viruses in pig manure following

mesophilic anaerobic digestion. The authors detected viruses during the first 5 months of

the study. However, the authors did not report virus concentrations, virus reduction rates,

nor the HRT of the digester. Treatment of dairy manure wastewater by fixed-film

anaerobic digestion at a 3 day HRT and ambient temperature (280C), can achieve a 1

logo reduction of bacteriophages and viruses (Barnes, 2002, Davis, 2001).

Previous studies on viral inactivation by anaerobic digestion point reductions to

operational parameters (i.e., temperature and retention time). However, antagonism by

indigenous manure microflora has been suggested to play a critical role in inactivating

viruses during anaerobic digestion (Deng and Cliver, 1995, Ward, 1982). Therefore, the

role of indigenous microflora in reducing virus during anaerobic digestion should be

investigated.

Purpose

The purpose of the current study was to determine if the reduction of viruses in

flushed dairy manure wastewater during fixed-film anaerobic digestion at ambient

temperature is attributed to the presence of indigenous manure microflora.

Materials and Methods

Sample Collection, Characterization, and Preparation

Flushed dairy manure wastewater (influent) and anaerobically treated flushed dairy

manure wastewater (effluent) were collected from the University of Florida Dairy

Research Unit (DRU) in Hague, FL and characterized as previously described (see

Chapter 3)









Bacteriophages

Bacteriophages and their respective hosts were MS2 (ATCC 15597-B1) and E. coli

C-3000 (ATCC 15597), DX174 (ATCC 13706-B5) and E. coli (ATCC 13706), and

PRD1 and Salmonella Typhimurium (ATCC 19585).

Experimental Design

Whole and soluble fractions of influent and effluent were prepared as previously

described (see Chapter 3). Soluble fractions were inoculated with each organism. The

samples were transferred to the anaerobic chamber and incubated for 3 days at 280C.

Groundwater collected from the DRU was used as a negative control for experiments

using bacteriophages.

Bacteriophage Quantification

Bacteriophages were enumerated as plaque forming units per ml (PFU/ml) using the

soft agar overlay technique (Hurst, 1997).

Anaerobic Conditions

Anaerobic conditions during incubation were maintained in an anaerobic chamber as

previously described (see Chapter 3).

Statistical Analysis

Log1o increases or decreases were determined under each condition by subtracting

the final loglo transformed concentration from the initial loglo transformed concentration.

Each condition was compared with the positive control and negative control using one-

way ANOVA and Duncan's Method with a significance level of 0.05. The statistical

analysis was performed using ProStat v 3.5.









Results

Wastewater Characteristics

Characteristics of the wastewaters used for experiments are given in Chapter 3 (see

Table 3-2).

Influence of Whole and Soluble Fractions of Wastewater

To determine if the indigenous microflora affected the concentrations of

bacteriophages, influent and effluent with and without indigenous microflora (i.e., whole

and soluble fractions, respectively) were inoculated with approximately 105 PFU/ml of

bacteriophages. The samples were incubated under anaerobic conditions for 3 days at

280C. In the soluble fractions of influent and effluent, bacteriophages (MS2, PRD1 and

OX174) were stable throughout the duration of the experiment, and were not

significantly different (P > 0.05) from those observed in groundwater (Table 5-1).

However, when incubated in the presence of indigenous microflora, there were

significant reductions (P < 0.05) in the concentrations of each bacteriophage in both

influent and effluent (Table 5-1). There was a significantly higher reduction (P < 0.05)

of MS2 and OX174 in effluent than in influent. The highest reductions were observed

with PRD-1. However the reductions of PRD1 observed in influent and effluent were not

significantly different (P < 0.05). These results show that the activity of indigenous

microflora in influent and effluent can contribute to bacteriophage inactivation under

anaerobic conditions and ambient temperature.

Discussion and Conclusions

The current study demonstrates the role of indigenous microflora on inactivation of

viruses during anaerobic digestion. All viruses studied were stable in soluble fractions of

wastewater incubated under anaerobic conditions at 280C for 3 days. However, in the









presence of indigenous microflora, all the viruses studied were reduced by greater than 1

logo.

Previous studies have shown inactivation of viruses during mesophilic and

submesophilic (15 to 250C) temperatures may be attributed by microbial antagonism.

Pesaro et al (1995) conducted field studies to investigate virus inactivation in liquid

cattle manure stored under anaerobic conditions using membrane sandwiches with and

without pores. At submesophilic temperatures, decimation times for viruses were

significantly lower for membranes with pores than those without. The results suggest

that microbial products may contribute to inactivation of viruses. Ward (1982)

investigated antiviral activity of microorganisms in activated sludge (mixed-liquor

suspended solids MLSS) against poliovirus 1 under aerobic conditions. The study

demonstrated that viruses were inactivated in the presence of MLSS and in broth

previously incubated with MLSS. These results suggested that microbial products could

contribute to inactivation of viruses during anaerobic treatment. Deng and Cliver (1992)

found similar inactivation of poliovirus 1 by bacteria in unmixed, stored swine manure.

The authors reported that decimation times in manure incubated under aerobic conditions

at 37C and 25C were significantly less than those in cell-free controls at the same

temperatures. A later study by the same authors suggest that microbial mediated

inactivation of viruses may not always be enzymatic (Deng and Cliver, 1995).

Production of antiviral substances that are not enzymatic (virolytic substances) has been

demonstrated with Pseudomonas aeruginosa (Cliver and Herrmann, 1972).

The current study provides further evidence of the critical role indigenous

microflora have during fixed-film anaerobic digestion. Although microbial processes









during anaerobic digestion are shown to reduce virus concentrations, complete removal is

not achieved. Reductions of greater than 3 loglo may be achieved, but as demonstrated

by previous studies either longer retention times or higher operating temperatures are

necessary to allow for more reduction. However, operations desiring to minimize energy

input to digesters must balance performance with wastewater production, thereby

sacrificing extensive virus removal. Therefore, effluents from digesters will inherently

contain residual concentrations of active viruses, which may be introduced to the

environment during land application.









Table 5-1 Impact of indigenous microflora on viruses suspended in groundwater and
wastewater.

Logio Reduction
Organism Groundwater Influent Effluent
Soluble Whole Soluble Whole Soluble Whole
fraction fraction fraction fraction fraction fraction

MS2 -0.5B -0.5a -0.2A -0.8b 0.0 -1.3b

PRD1 -0.3A -0.3a -0.1A -2.5b -0.2^ -2.4b

(DX174 -0.5A -0.5a -0.2A -1.2b -0.3A -1.8c



Notes: Groundwater, influent, and effluent were prepared as described in the text (see Chapter 3).
Samples were inoculated with viruses and incubated under anaerobic conditions at 280C for 3 days. Each
value is an average logo10 reduction (n = 3).
A-B, a-c Values with the same letter within the same row are not significantly different (P < 0.05).














CHAPTER 6
TRANSPORT OF VIRUSES IN SOIL AMENDED WITH ANAEROBICALLY
DIGESTED FLUHSHED DAIRY MANURE WASTEWATER

Introduction

Intensification and concentration of large dairy operations have led to issues with

manure management. Two major concerns are nuisance odors and possible

contamination of groundwater resources following land application of manure. In an

attempt to reduce these problems, a fixed-film anaerobic digester has been constructed at

the University of Florida's Dairy Research Unit (DRU). Freestall barns at the DRU are

hydraulically flushed with water to remove animal manure. The fixed-film digester then

treats the flushed dairy manure wastewater (influent) before it is land applied for forage

crop production. Anaerobic digestion significantly reduces the COD of flushed dairy

manure (Wilkie, 2005). Previous studies have shown that this unit can also reduce the

levels of indicator and pathogenic bacteria and bacteriophages by approximately 90%

(Davis et al., 2001). However, the use of fixed-film anaerobic digesters on flushed

manure is a relatively new technology and the fate of viruses following land application

of anaerobically digested flushed dairy manure (effluent) is unknown.

Virus Inactivation during Anaerobic Digestion

Viruses are known to be associated with and survive in animal manure (Elliott and

Ellis, 1977, Mawdsley et al., 1995, Pesaro et al., 1995). However, anaerobic treatment

has been shown to inactivate viruses. Anaerobic digestion at ambient temperature (25 to

31C) and a 3-day HRT has been shown to achieve a 1.5 logo reduction of somatic and









male-specific bacteriophages from flushed dairy manure wastewater (Davis et al., 2001).

Lund et al.(1996) found that mesophilic (350C) anaerobic digestion of swine and cow

liquid manure required 9 days to achieve a 4 logo reduction (99.99%). Under

thermophilic (55C) conditions, the system required 6 days to achieve a 4 logo reduction

of porcine parvovirus. Anaerobic digestion is effective for reducing the load of viruses in

animal manure before land application. However, residual viral concentrations can be

detected in effluents of anaerobic systems treating animal manure and be detected in soil

where wastewater has been applied (Derbyshire, 1976, Derbyshire et al., 1986) Although

anaerobic digestion achieves significant reductions of viruses from animal manure,

residual concentrations of viruses can potentially lead to groundwater contamination

following land application of effluents.

Viral Attachment to Soil

Viruses in water utilize two mechanisms for attaching to soil, hydrophobic and

electrostatic interactions. Physical and chemical parameters that affect virus adsorption

to soil include the pH of the aqueous media and the soil, flowrate through the soil matrix,

soil organic matter, percent clay, and ionic strength of the suspending medium (Goyal

and Gerba, 1979, Kinoshita et al, 1993, Lance et al., 1976, Zhuang and Jin, 2003).

pH and isoelectric point. Previous studies have investigated transport,

adsorption, and survival of viruses in wastewater following land application of

wastewater. Several environmental factors have been found to influence viral adsorption

in soil. Organic matter and exchangeable iron are inversely correlated with viral

adsorption to soil (Gerba and Goyal, 1981, Hurst et al., 1980). However, one of the most

important factors is pH (Goyal and Gerba, 1979). In general, an increase in soil pH will

result in a decrease in viral adsorption, whereas a decrease in soil pH will increase viral









adsorption. However, environmental factors, including pH, do not influence the

adsorption of all viruses to the same degree. Gerba and Goyal (1981) examined the

differences in adsorption among viruses and categorized the viruses into two groups

based on factors that contributed to adsorption. The factors that were most important for

adsorption of group I viruses, which included OX 174 and MS2, to soil were pH, organic

matter content, and exchangeable iron. However, group II viruses, which included

poliovirus 1 LSc and coxsackie B3, soil particle surface area was the only significant

variable that contributed to adsorption.

The impact of pH on viral adsorption is related to the isoelectric point (pI) of the

virus (Dowd et al., 1998). The isoelectric point is the pH at which the virus has no net

electrical charge. In general, viral adsorption will occur in soils with a pH lower than that

of the pI of virus. Viruses and soil particles generally have a net negative charge at a

neutral pH. At low pH values, viruses acquire a net positive charge, resulting in

adsorption to soil. At high pH values, viruses acquire a net negative charge and

adsorption to soil is minimal and results in elution or desorption of the virus from the

soil. The adsorption effects observed with viruses are the result of alterations to the

proteins on the surface of the virion during pH changes; whereas changes to the charge

on soil particles are minimal (Berg, 1987, Dowd et al., 1998).

Hydrophobic interactions. The side chains of several amino acids found on the

protein coat of viruses are nonpolar and attract other nonpolar compounds or groups.

Hydrophobic bonding occurs when nonpolar groups aggregate. Viruses with coats that

are predominately composed of hydrophobic proteins will tend to use hydrophobic

interactions. Such viruses include MS2 and echovirus 5 (Shields and Farrah, 2002).









Futhermore, viruses that contain host-derived phospholipid membranes, such as PRD1,

will also utilize hydrophobic bonding (Hanninen et al., 1997). Hydrophobic interactions

may be demonstrated when the pH of an aqueous medium is adjusted to the pI of the

virus. At that point, hydrophobic interactions may predominate (Berg, 1987).

Electrostatic interactions. Electrostatic interactions occur between charged side

chains of amino acids on viral protein coats and charges on surfaces. Shields and Farrah

(2002) compared viral adsorption by electrostatic and hydrophobic interactions. Using

DEAE-Sepharose, a solid with charged groups, virus adsorption was characterized by

elution from the solids with solutions increasing in ionic strength. Several viruses,

including poliovirus 1 LSc and T4, were shown to utilize strong electrostatic interactions.

In aqueous media, such as natural waters, the pH is usually neutral and above the pI of

most viruses. In such instances, the charge of the viron and soil matrix will be the same

and electrostatic interactions will depend on the concentration of cations present with in

the aqueous media or the soil. Cations may interact with both the viron and soil surface

generating a salt bridge, resulting in adsorption (Berg, 1987).

Purpose

Viruses have been shown to be inactivated during anaerobic digestion (Davis et al.,

2001, Lund et al., 1996). However, residual concentrations of viruses may be introduced

to the environment during land application of treated wastewater. To date, the adsorption

of viruses to soil following land application of effluent from a fixed-film anaerobic

digester treating flushed dairy manure wastewater has not been studied. The purpose of

the current study is to investigate the adsorption and survival of viruses in flushed dairy

manure wastewater and in anaerobically digested wastewater to soil.









Materials and Methods

Collection and Analysis of Soil Samples

Soil was collected from the DRU in Hague, FL, in an area mapped as typic

Quartzipsamments; sandy soil with greater than 90% having particle size between 0.02 to

2.0 mm (USDA, 1999). Samples were taken at 0.8 m depth from a sprayfield that

receives effluent from a tertiary short-term storage pond. Soil samples were thoroughly

mixed to yield a composite sample. The soil was air dried at room temperature and

sieved. Batch adsorption studies were performed on soil with particle size less than 0.2

mm. To increase the flow rate through columns, soil with particle size of 0.2 to 0.8 mm

was used for column studies. Soil pH and conductivity were measured as described by

Mylavarapu and Kennelley (2002). Soil particle size analysis was conducted on the

whole, batch, and column soil samples.

Collection and Analysis of Wastewater, Groundwater, and Rainwater Samples

One liter samples of influent and effluent were collected from the DRU manure

management facility. Grab samples of influent were collected from a wet well prior to

anaerobic treatment. Effluent was collected from a port located on the fixed-film

anaerobic digester. After settling, supernatant fractions of both wastewaters were used

for experiments. Groundwater was collected from wells located at the DRU and

rainwater was collected during storm events. Sample pH and conductivity were

measured according to standard methods (Standard Methods, 1998).

Viruses and Viral Assays

Bacteriophages and their hosts used in this study were MS2 (ATCC 15597-B1),

host E. coli C-3000 (ATCC 15597); 4X174 (ATCC 13706-B5), host E. coli (ATCC

13706); and PRD1, host Salmonella Typhimurium (ATCC 19585). Phages were









quantified as plaque-forming units (PFU) using the agar overlay technique, as described

by Hurst (1997). Poliovirus 1 LSc (ATCC VR-59) was assayed as PFU on Buffalo green

monkey kidney cells, as described by Hurst (1997). Characteristics of the viruses used in

the study are given in Table 6-1.

Virus Stability

The stability of viruses in groundwater and wastewater was tested. During all

batch and column studies, viruses were suspended in groundwater and wastewater and

assayed at the beginning and end of each experiment to check for inactivation during an

experiment. Viral assays were performed as previously described.

Attachment and Detachment Studies

Batch studies. Batch adsorption studies were performed with groundwater,

influent wastewater, and effluent from the anaerobic digester. The study was designed

using nine batches so that each experimental condition was performed in triplicate. The

entire experiment was performed twice to yield a total of six observations for each

experimental condition. Two ml samples of groundwater, influent, or effluent were

inoculated with viruses and added to 1 g soil samples (particle size < 0.2 mm). Samples

were mixed on a reciprocating shaker at 55 rpm for 1 h, followed by centrifugation at

3,000 x g for 10 min. The supernatant fraction was collected and assayed. The soil

pellets were mixed with 10 ml 3% beef extract (BE), pH 7, for 30 min to recover

adsorbed viruses (Hurst, 1997). The samples were centrifuged again and the supernatant

fraction was collected and assayed for viruses as previously described.

To determine the influence of groundwater, influent, and effluent on desorption of

enteroviruses and bacteriophages from soil, 1 g soil samples were set up as previously

described. Each soil sample was mixed with 2 ml of inoculated groundwater for 1 h and









centrifuged as previously described. The supernatant fraction was assayed and the soil

pellets were treated with 10 ml of either groundwater, 3% BE, influent, or effluent for 30

min. The samples were centrifuged at 3,000 x g for 10 minutes and the supernatant

fractions were assayed

Column studies. Adsorption studies with soil columns were performed using 45 g

of soil (0.2 to 0.8 mm) packed in 60 ml syringes with a borosilicate fiberglass filter in the

bottom of the syringe. Each experimental condition was performed in triplicate. One

pore volume (20 ml) of groundwater, influent, or effluent was inoculated with viruses and

passed through the columns at 1 ml/min. The percolate was collected and assayed to

determine the percentage of viral adsorption. Each column was then removed from the

syringes and placed in a 500 ml centrifuge bottle. From each column, 5 g was removed,

dried and then mixed with 10 ml of deionized water for 2 h to measure soil pH. The

remaining 40 g of soil was mixed for 30 min with 40 ml 10% buffered BE (100 g/L BE,

13.4 g/L sodium phosphate dibasic, 1.2 g/L citric acid) (Hurst, 1997). The samples were

centrifuged at 5,000 x g for 10 min and then the supernatant fraction was collected and

assayed.

To study potential mobilization of viruses by influent and effluent, one pore

volume of groundwater inoculated with viruses was added to each column. The percolate

was collected and assayed to determine the percentage of viral adsorption. Six pore

volumes of rainwater was added to each column. The percolates were collected and

assayed for viruses. An additional six pore volumes of either rainwater, influent, or

effluent was passed through the columns. The percolates were collected and assayed for









viruses. The columns were removed from the syringes and mixed with BE and assayed

for recovered viruses as described previously.

Attachment and Detachment Mechanisms

Soil COD retention. The amount of soluble organic retained by the soil,

expressed as soil soluble chemical oxygen demand (soil SCOD), was calculated by

subtracting wastewater initial SCOD from SCOD of the wastewater after percolating

through the soil column. The percentage of viruses mobilized at each pore volume was

plotted against soil-retained SCOD for the corresponding pore volume and analyzed by

linear regression.

Influence of pH on virus adsorption. A 100 ml sample of either groundwater,

influent, or effluent was adjusted to pH 3.5 with HC1. Phages were added to the adjusted

groundwater and wastewater samples. Inoculated samples (2 ml) were added to 1 g of

soil samples (< 0.2 mm) and mixed on a reciprocating shaker at 55 rpm for 1 h. The

samples were centrifuged, assayed, and the viruses were recovered as described

previously for batch studies.

Wastewater fractionation. Influent and effluent samples were fractionated using

cellulose ester dialysis tubing (Spectra/Por Biotech). Groundwater, influent, and

effluent samples (20 ml) were transferred to dialysis tubing with molecular weight cut-

offs (MWCOs) of 100 kDa, 10 kDa, and 1 kDa. Groundwater samples were dialyzed

against 1 L influent for 3 days at 40C. Influent and effluent samples were dialyzed

against 2 L deionized water for 3 days at 4C. A larger volume of deionzed water was

used for dialyzing wastewater samples to remove as much of the target sized compounds

as possible. The samples were removed and a 10 ml aliquot of each sample was









inoculated with viruses. Adsorption to soil, virus assays, and virus recovery was

performed as previously mentioned for batch studies.

Influence of detergents on adsorption and desorption. Adsorption studies were

performed using a cationic detergent (hexadecyltrimethylammonium bromide, HTAB,

Sigma), anionic detergent (sodium dodecyl sulfate, SDS, FisherBiotech) and a nonionic

detergent (polyoxyethylene sorbitan monooleate, Tween 80, Fisher Scientific) were

added to groundwater, influent, and effluent samples to a final concentration of 0.01%.

The samples were inoculated with viruses and adsorption, assay, and recovery was

performed as previously described for batch studies.

Batch soil samples (1 g, particle size < 0.2 mm) were pretreated with groundwater

supplemented with HTAB, SDS, or Tween 80 to a final concentration of 0.01% to

determine the impact on virus adsorption to soil. Batch samples were mixed with 10 ml

of groundwater detergent solution for 1 h on a reciprocating shaker table as previously

mentioned. The samples were centrifuged as described earlier for batch studies and the

supernatant was discarded. The pellet was washed three times with 10 ml groundwater to

remove any detergent not retained by the soil. To the final soil pellets, 2 ml of

groundwater inoculated with viruses was added. The samples were mixed, centrifuged,

assayed, and the viruses were recovered as previously described for batch studies.

The influence of detergents on desorption of viruses attached to soil samples was

performed by adsorbing viruses in groundwater to soil as previously described for batch

soil adsorption studies. Groundwater was supplemented with HTAB, SDS, or Tween 80

to a final concentration 0.01%. The soil samples with attached viruses were mixed with

10 ml of detergent solution for 30 min, centrifuged, and assayed as previously described









for batch soil studies. A secondary elution was performed with 3% BE as previously

described for batch soil studies.

Virus Survival in Soil

The survival of viruses in groundwater and wastewater attached to soil was

determined. Groundwater, influent, and effluent were inoculated with MS2, PRD1,

OX174, and poliovirus 1 LSc at 1 x 107 PFU/ml. Inoculated solutions were added to soil

as previously described for batch soil studies. Samples were set up in triplicate for each

time point. Once a week, viruses were recovered with 3% BE and assayed as previously

described for batch soil studies.

Risk Assessment of Flushed Dairy Manure

A risk assessment was performed to determine the probability of infection

following exposure to groundwater where influent and effluent was applied to land. The

following exponential dose-response model was used to estimate the risk of infection:

7C = 1- 10-(d/k)

where, n is the probability of infection, d is the amount of exposure to the organism, and

k is the infective dose (Rose and Hass, 1999). Infectious dose data for adenovirus

(PHAC, 1999), rotavirus (Graham et al, 1987), and Norwalk virus (Schaub and Oshiro,

2000) was used for the risk assessment.

Statistical Analysis

The percentage of viruses eluted was calculated by dividing the amount of viruses

in the eluent by the amount of viruses adsorbed to soil. The mean percentages of viruses

eluted and the differences in viral adsorption between natural and detergent-pretreated

conditions were analyzed by Student's t-test with a significance level of 0.05. A









comparison of the percentage of viruses adsorbed in groundwater and various virus

characteristics (i.e. isoelectric point, type of interaction, and structure) was subjected to

regression analysis to determine correlations. Regression analysis was also used to

determine the effect of soil SCOD retention on virus mobilization in soil columns. All

analysis was performed using ProState v3.5.

Results

Soil Characteristics

The soil used in the batch and column experiments differed in composition (Table

6-2). Soil used for batch and column studies differed in the amount and type of sand.

The amount of silt and clay, the reactive species in soil, was higher in the soil used for

batch studies than in the column studies.

Batch Studies

The adsorption of viruses in groundwater, influent and effluent to soil are shown in

Figure 6-1. When the viruses were suspended in groundwater, there was greater

adsorption of I X174 (> 99 %) and poliovirus 1 (96 %) than of MS2 (72 %) and PRD1

(48 %) (Table 6-3). In influent, adsorption decreased to 17 % for MS2 and < 1 % for

PRD1. The presence of influent also decreased the adsorption of 4X174 (78 %), but had

little effect on the adsorption of poliovirus 1 (93 %). The adsorption of MS2 (45 %) and

PRD1 (8 %) was less in effluent than in groundwater, but greater than in influent. The

adsorption of 4X174 and poliovirus 1 was similar in influent and effluent. These results

indicate that the adsorption of viruses was influenced by the presence of effluent and

influent and by the type of virus.

The phages adsorbed to soil in the presence of groundwater were desorbed by

influent (Table 6-4). The percent adsorption to soil observed for MS2 and PRD1 in









groundwater was lower than for initial adsorption experiments. This difference may be

due to variable soil composition during subsampling. The percentage of all phages

detached by influent was significantly higher (P < 0.05) than the percentage detached by

effluent or groundwater. The percent eluted by either effluent or groundwater was not

significantly different (P < 0.05) for MS2 and PRD1. Poliovirus 1 adsorbed to the soil

was not eluted by groundwater, influent or effluent. These results show that, except for

poliovirus 1, untreated wastewater can detach more adsorbed phages from soil as

compared with anaerobically treated wastewater.

Column Studies

The adsorption of viruses in groundwater, influent and effluent to soil columns is

shown in Figure 6-2. The adsorption pattern for the viruses studied was similar to that

observed in the batch studies in that the adsorption of t X174 (97 %) and poliovirus 1 (99

%) in groundwater was greater than the adsorption of MS2 (88 %) and PRD1 (54 %). In

contrast with the batch studies, the difference between adsorption of MS2 and PRD1 in

the presence of influent and effluent was small.

As observed in the batch studies, influent was found to mobilize adsorbed viruses

(Table 6-5). Mobilization of MS2, PRD1 and I X174 by influent was significantly

higher (P < 0.05) than by effluent or rainwater. Influent mobilized 2 % of poliovirus 1,

whereas no mobilization was observed with effluent or rainwater.

Conductivity and pH

The initial pH of the soil samples was considerably lower than the pH of

groundwater, rainwater, influent and effluent samples (Table 6-6). In both the batch and

column experiments, there was an increase in pH in all samples following mixing of soil

with one of the solutions described above. However, we found that normalizing the pH









of the soil to pH 7.0 during the experiment did not affect adsorption and elution (Table

6-7). Furthermore, during percolation, conductivity, or ionic strength, of the soil samples

decreased with rainwater and increased with influent and effluent (Table 6-6).

Attachment and Detachment Mechanisms

Soluble organic. MS2 elution displayed a positive correlation with soil SCOD (r

= 0.936) (Figure 6-3). PRD1 (r = 0.103) and
low correlation with soil SCOD.

Wastewater fractionation. To determine the size of the compounds) in influent

and effluent affecting virus adsorption to soil, dialysis was used to remove compounds

smaller than 100 kDa, 10 kDa, and 1 kDa from the wastewaters. Viruses were inoculated

into the wastewater samples that retained compounds larger than the indicated MWCOs.

In samples of influent where molecular weight fractions larger than 10 kDa were

retained, adsorption of MS2 (Figure 6-4) was similar to adsorption in groundwater for

batch studies (Figure 6-1). This result suggests that compounds in the influent that were

smaller than 10 kDa were mainly responsible for interfering with adsorption of MS2 to

soil. Adsorption did not significantly increase in the influent where compounds larger

than 1 kDa were retained. However, adsorption of MS2 to soil was higher in the effluent

where compounds larger than 1 kDa were retained, as compared with adsorption with

groundwater in batch studies. These results suggest that in the influent, removal or

alteration of compounds between 10 kDa and 1 kDa interferes with adsorption of MS2 to

soil.

The adsorption of PRD1 to soil was impacted by different fractions of influent and

effluent (Figure 6-5). In the effluent, retention of compounds larger than 10 kDa did not









interfere with adsorption of PRD1 to soil. In the influent, retention of compounds larger

than 100 kDa did not interfere with adsorption of PRD1 to soil. These results suggest

that compounds in the influent, between 100 kDa and 10 kDa, affect adsorption ofPRD1

to soil.

The adsorption pattern of OX174 in fractionated wastewater to soil is shown in

Figure 6-6. In both the influent and effluent, retention of compounds larger than 100 kDa

did not interfere with adsorption of ODX174 to soil. There were no significant differences

observed in adsorption between native wastewater and wastewater retaining compounds

larger than 10 kDa and 1 kDa. This finding suggest that compounds in the influent,

between 100 kDa and 10 kDa, interfere with adsorption of OX174 to soil.

Influent compounds smaller than 100 kDa and 10 kDa were transferred to

groundwater, by dialyzing 20 ml of groundwater with 100 kDa and 10 kDa MWCOs

against 1 L of influent Viruses were inoculated into groundwater, dialyzed groundwater,

influent, and influent used for dialysis. The samples were mixed with soil as previously

described for batch studies. Adsorption patterns for PRD1 and OX174 are shown in

Figure 6-7. Adsorption of PRD1 and OX174 to soil decreased when suspended in

groundwater containing influent compounds smaller than 100 kDa. Adsorption of PRD1

in influent and groundwater dialyzed with influent to soil was similar. Adsorption of

0X174 to was lower using suspensions of groundwater dialyzed with influent than in raw

influent. Furthermore, a slight increase in adsorption was observed for both viruses

suspended in influent used for dialysis. These results, along with the results from Figures

6-5 and 6-6, demonstrate the interference of influent compounds smaller than 100 kDa on

adsorption of PRD1 and OX174 to soil.









The influence of influent compounds smaller than 10 kDa on adsorption of MS2 to

soil was also performed. MS2, suspended in groundwater containing influent compounds

smaller than 10 kDa, was not detected in the supernatants after mixing with the soil for 1

h. Furthermore, MS2 was not detected in the initial stock solutions of groundwater

containing influent compounds smaller than 10 kDa. To determine if influent compounds

smaller than 10 kDa inactivated MS2, groundwater and groundwater dialyzed against

influent was inoculated with MS2. A 300 il aliquot was taken at 10 min intervals for 1

h. Influent compounds smaller than 10 kDa were found to inactivate MS2 (Figure 6-8).

Inactivation was not observed in experiments using the whole fraction of influent. This

may be due to interaction between particulate organic and the compounds causing

inactivation, thereby preventing inactivation of MS2.

Influence of isoelectric point on virus adsorption to soil. Adsorption of viruses

in groundwater to soil in batch studies correlated with isoelectric point (r = 0.973) (Figure

6-9).

Influence of pH on virus adsorption to soil. To determine the effect wastewater

had on virus adsorption at pH values below the pI, the pH of the groundwater and

wastewaters were adjusted to 3.5. Adsorption to soil by all three viruses in groundwater

and wastewater adjusted to pH 3.5 increased to > 99 % (Table 6-8).

Hydrophobic and electrostatic interactions. Viruses utilize hydrophobic and

electrostatic interactions to bind to soil. To determine the type of interactions used by the

viruses under study, ionic and nonionic detergents were used. Ionic and nonionic

detergents were added to groundwater and wastewater to a final concentration of 0.01 %,

the maximum concentration where viruses remained active. The detergent solutions were




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FIXED-FILM ANAEROBIC DIGESTION: MECHANISMS OF PATHOGEN REDUCTION AND IMP ACTS ON VIRUS ADSORPTION TO SOIL By JOHNNY A. DA VIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Johnny A Davis

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To Ethan and Joshua.

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ACKNOWLEDGMENTS I would like to thank Dr. Samuel R. Farrah, chair of the supervisory committee, for providing support, research facilities, and guidance towards completion of this body of work. I would like to extend a sincere appreciation to the co-chair of the supervisory committee, Dr. Ann C. Wilkie. Her constant persistence and guidance pushed the work forward to completion. Through our numerous scientific discussions and constant mental challenging, I have gained a tremendous appreciation for my field of study and the pursuit of knowledge in general. I have no doubt that our interactions will help me develop a successful career as an independent scientist. I thank the remaining members of the supervisory committee, Dr. Ben Koopman, Dr. Peter Kima, and especially, Dr. Madeline Rasche, for providing valuable insight to career development. I thank my wife, Nicole Davis, for her patience and support during this long journey. I would like to thank my parents, Harvey Moffitt and Emma T Davis, for their wisdom and encouragement. I would also like to thank Dr. Anne Donnelly for being a mentor and friend. I would also like to acknowledge David Armstrong and the Dairy Research Unit staff for their help during various stages of the project. I would like to thank the Department of Microbiology and Cell Science faculty and staff. I also extend an appreciation for the McKnight Doctoral and Southeast Alliance for Graduate Education and the Professoriate Programs for providing funding during my graduate career. Lastly I would like to acknowledge my reunion with my biological parents Namon and Debbie Martin and the introduction to the family I never had the opportunity lV

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to know. During the last four years I have enjoyed learning about my family and I look forward to our future together as we continue to close the 24-year gap. V

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TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES .......................... .................................................................................. ix LIST OF FIGURES ........................................................................................................... xi ABSTRACT ...................................................................................................................... xii CHAPTER 1 INTRODUCTION ........................................................................................................ 1 2 PATHOGENIC ORGANISMS IN BOVINE MANURE AND THEIR REDUCTION BY ANAEROBIC DIGESTION ...... ... ... ........ .................. ....... ... .......... 4 Pathogenic Organisms in Dairy Cattle Manure ........................................................... .4 Zoonotic Bacteria .......................... .. .......... .......................................................... 5 Mastitic Bacteria .................................................................................................. 10 Pathogenic Viruses ............................ ........ ... ..................................... ... ..... ..... .... 12 Parasitic Protozoa ............ ............... . ... ............................................................. 14 Anaerobic Digestion and Pathogenic Organisms ...... .... .... ....................................... 15 Anaerobic Digestion ..... ................... .... ..... ... ........... ... ....... ... ................ . .......... ... 15 Anaerobic Digesters .............. .............. .. .... .... ... ................................................. 16 Microbial Diversity in Anaerobic Digesters ..... ........... ..... ..... .. .............. .......... ... 18 Pathogen Decimation during Anaerobic Digestion of Animal Manure ... ....... .... 20 Summary ........................ ... . ....... ...... . ........................ .... ...... ................... .................. 27 Purpose of Study ........ ... ...... ...... ............ ..................... ..... .......................................... 28 3 INDICATOR AND PATHOGENIC BACTERIA REDUCTION BY ANAEROBIC DIGESTION: THE ROLE OF MICROBIAL COMPETITION AND SUBSTRATE LIMITATIONS ........................... ...................................... ....... 29 Introduction ........ ...... .............. . ..... ......... ... ........... .............................. .. ..... .... ..... .... 29 Purpose ................................................. ..... ....... . ... ...... .. ... .. ....................... ........ ...... 31 Materials and Methods ................... ....... . ...... ................................................ ....... . ... 32 Dairy Research Unit ...... ...................... ..... ....... .......... .. ...................... ... ............ 32 Manure Handling ............................ . ............. ...... ... ................ ..... ..... .... ............. 32 V I

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Fixed-film Anaerobic Digester ........................................................................... .32 Sample Collection, Characterization, and Preparation ....................................... .33 Bacterial Cultures ................................................................................................ 34 Bacterial Quantification ..................................... ........................................... ..... 34 Anaerobic Conditions ......... .... . ........................................................................... 35 Survival in Whole and Soluble Wastewater Fractions ................ ....................... .35 Inhibition and Wastewater Supplementation Studies ......................................... .36 Statistical Analysis .............................................................................................. 37 Results ......................................................................................................................... 37 Wastewater Characteristics ................................................................................. 3 7 Growth of Bacteria in Whole and Soluble Fractions of Wastewater ................. .38 Total aerobic, anaerobic, and facultative bacteria ....................................... .38 Escherichia coli and fecal coliforms . ...... ...... ......... ........... .... ...................... 39 Enterococcus spp .............................. ..... ....................................................... 40 Salmonella spp .......................... .... ....................... ... ............. ................ ...... 41 Escherichia coli 0157:H7 ........................................................................... .41 Staphylococcus aureus .......... ..... .......... ... ..................................................... 41 Discussion and Conclusions . ................... ......... ..... ...................................... ............ 43 4 INDICATOR AND PATHOGENIC BACTERIA REDUCTION DURING ANAEROBIC DIGESTION: ATTACHMENT TO THE FIXED-FILM ................ 56 Introduction ..... ... .............. ... ................................ ...................................... ........... ...... 56 Purpose ....................................................................................................................... 56 Materials and Methods .................... ... .................................................... ..... ............. 57 Pilot-Scale Fixed-Film Reactors .......................................................................... 57 Survival Comparability with Indigenous Fecal Coliforms ...................... ... ...... ... 57 Bio film Attachment Studies .................... ..... ......... ... ...................... .... ..... .......... 58 Biofilm Sampling and Examining ........ ........ ................ ......... ............ .... ........ ..... 58 Results ............. . ... ..... ................ ...................... ..... ............................................. .......... 59 Fecal Coliform and GFP E. coli Survival.. .... .............. ..................... ......... ... ... .... 59 Bio film Attachment Studies ................... ..... . ...................................................... 59 Discussion and Conclusions .................... .................................................................. 60 5 BACTERIOPHAGE REDUCTION DURING ANAEROBIC DIGESTION: THE ROLE OF INDIGENOUS MICROFLORA ................. .... .............................. ..... .... ... 66 Introduction ............................................ ... . ... ...... ............... . .... ... .............................. 66 Purpose ..... ... ....... ........... ......... ....... .............. ............................................... ......... ... 67 Materials and Methods .................................... ....................................................... ... 67 Sample Collection, Characterization, and Preparation .................... . .................. 67 Bacteriophages ........ ...... .... ................ ................................... ....... ....... ..... .......... 68 Experimental Design ............ ......... ....... .... ... ......... ..... .................. ....................... 68 Bacteriophage Quantification ........ ... ........ .... ................ ........... ........................... 68 Anaerobic Conditions .................. .............. .. ........................................................ 68 Statistical Analysis .............. ......... ...... ..... .............................. ..... ........................ 68 Results ......... . ................................. ..... ............ ......... ................................. ........ ....... 69 Vll

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Wastewater Characteristics ................................................................................. 69 Influence of Whole and Soluble Fractions of Wastewater .................................. 69 Discussion and Conclusions ....................................................................................... 69 6 TRANSPORT OF VIRUSES IN SOIL AMENDED WITH ANAEROBICALLY DIGESTED FLUHSHED DAIRY MANURE WASTEWATER .............................. 73 Introduction ................................................................................................................. 73 Virus Inactivation during Anaerobic Digestion .................................................. 73 Viral Attachment to Soil. ..................................................................................... 74 Purpose ............................................................................ .......................................... 76 Materials and Methods ............................................................................................... 77 Collection and Analysis of Soil Samples ............................................................ 77 Collection and Analysis of Wastewater, Groundwater, and Rainwater Sainples ............................................................................................................ 77 Viruses and Viral Assays ..................................................................................... 77 Virus Stability ...................................................................................................... 78 Attachment and Detachment Studies ................................................................... 78 Attachment and Detachment Mechanisms ..... .................................................... 80 Virus Survival in Soil .......................................................................................... 82 Risk Assessment of Flushed Dairy Manure ........................................................ 82 Statistical Analysis ............. .... ...... ..... ................................... ....... ...................... 82 Results . .......................................... .. .... .. ..................................................................... 83 Soil Characteristics ......... .... ................... . ......................................................... 83 Batch Studies ....................................................................................................... 83 Column Studies ............................................... ................................................... 84 Conductivity and pH ........................................ ................................................... 84 Attachment and Detachment Mechanisms .......................................................... 85 Risk Assessment of Viruses Following Land Application .................................. 90 Survival of Viruses in Soil .................................................................................. 90 Discussion and Conclusions ............................................................... ... ..................... 90 7 SUMMARY OF CONCLUSIONS ................................................................ .. ......... 122 LIST OF REFERENCES .............................................................................. .. ................. 125 BIOGRAPHICAL SKETCH ................................. .. ........................................................ 140 Vlll

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LIST OF TABLES 3-1. Percentage of carbon from various sources in yeast extract.. ................................... .49 3-2. Characterization of samples used for all experiments ............................................... 50 3-3. Growth of indicator and pathogenic bacteria in the soluble fraction of wastewater ............................................... ........ .. ......... ............................................ 51 3-4. Growth of indigenous indicator bacteria and S. aureus in the whole fraction of wastewater. ............................................................................ .... ............................. 52 3 5. Comparison of growth between indigenous bacteria in wastewater at 38C and 28C . ...................................................................................................... ..... ........... 53 3-6. Determination of inhibitory or nutritional limitation on the growth of indigenous S. aureus .... .......... .................. .......... ....................................................... ................ 54 37. Growth of S. aureus in soluble effluent and soluble effluent with various amendments .................................................................... ....................... .. ................ 55 5-1. Impact of indigenous microflora on viruses suspended in groundwater and wastewater ......................... . .. .... ........ ... .. ..... . ... ... .. .. .......................... ............... 72 6-1. Characteristics of viruses used in the study ....... ........................ ..... .......... ............. 97 6-2. Characteristics of test soil ............................................................... .......... ................ 98 6-3. Adsorption of Viruses in groundwater and wastewater to soil.. .. ............ ...... ..... ... 99 6-4. Elution of viruses adsorbed to soil. ... .. . .................................... . ... ........ .............. 100 6-5. Elution of viruses adsorbed in soil columns ............................................. .............. 101 6-6. Conductivity and pH changes in soil during batch and column experiments ......... 102 67. Adsorption of viruses in groundwater and wastewater to soil adjusted to pH 7 .0 .. 103 6-8. Influence of pH on adsorption of viruses to soil. ................ ........... ... ...... .............. 104 6-9. Elution of viruses adsorbed to soil by detergents in groundwater ..... . . .. ....... . ..... 105 I X

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6-10. Recovery of viruses with 3% BE from soil pretreated with detergents ................. 106 6-11. Estimated risk of infection from exposure to groundwater following land application of wastewater ................. ....... . ............................................................. 107 X

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LIST OF FIGURES Figure 4-1. Survival of fecal coliforms and GFP producing E. coli in wastewater .................. 62 4-2. Passage of GFP E.coli through the pilot-scale fixed-film anaerobic digester ...... 63 6-1. Adsorption of viruses in groundwater and wastewater to soil. .. ............ ... .......... 108 6-2. Adsorption of viruses in groundwater and wastewater to soil columns .............. 109 6-3. Correlation between soluble chemical oxygen demand retained by the soil and MS2 detachment from soil columns ... ......... .............. .. ................ ............ 110 6-4. Adsorption of MS2 in fractionated wastewater to soil. .... .............. ..... ............... 111 6-5. Adsorption of PRDl in fractionated wastewater to soil. ..................................... 112 6-6. Adsorption of Xl 74 in fractionated wastewater to soil. ........ ...... ...... ... .......... 113 67. Influence of influent compounds smaller than 100 kDa on adsorption of viruses to soil. ..... ................. ... ... ... .......................... .................... ................ ... 114 6-8. Inactivation of MS2 by wastewater compounds smaller than 10 kDa ...... .......... 115 6-9. Correlation between virus pl and virus adsorption to soil. .................. .... .......... 116 6-10. Adsorption of viruses in groundwater with 0.01 % final concentration of detergents to soil. ........... ..................................................... ..... ...... ....... ......... 11 7 6-11. Adsorption of viruses in influent with 0.01 % final concentration of detergents to soil. ................................. ... ... ........ ................................... ............ ........... ... 118 6 12. Adsorption of viruses in effluent with 0.01 % final concentration of detergents to soil. ............................... ......... ....... : .......... ............... .... ... .......................... .... 119 6 13. Adsorption of viruses in groundwater to soil pretreated with detergents ........ ... 120 6-14. Survival of viruses in soil. ........ ... .... .... .... . ... ................ ......... ..... ... ....... ......... 121 X I

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FIXED-FILM ANAEROBIC DIGESTION: MECHANISMS OF PATHOGEN REDUCTION AND IMP ACTS ON VIRUS ADSORPTION TO SOIL Chair: Samuel R. Farrah Cochair: Ann C. Wilkie By Johnny A. Davis August2006 Major Department: Microbiology and Cell Science Anaerobic digesters have been used for treatment of various wastewaters. Systems are usually operated at mesophilic (35C) temperatures and long hydraulic retention times (HRTs) (> 20 days). Pathogen reduction during anaerobic digestion is mainly correlated with operating temperature and HRT. Higher temperatures and longer HRTs result in increased rates of pathogen decimation. Previous studies show that a novel fixed-film anaerobic digester, operated at a low HR T t:; 3 days) and ambient temperature (~ .28C) treating flushed dairy manure wastewater, achieved significant reductions of indicator and pathogenic bacteria. Thus, we sought to determine factors that contributed to indicator and pathogenic bacteria reduction during operation at a low HRT and ambient temperature. The presence of indigenous microflora was found to reduce the proliferation of indicator and pathogenic bacteria. Bacteriophages wer e also inactivated by the presence of indigenous microflora. Also, decimation of Staphylococcus aureus was attributed to starvation, resulting from

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anaerobic digestion, while not suppressing the other test organisms to the same degree. Furthermore, attachment to the retained biofilm (i.e., fixed-film) contributes to reduction by removing indicator and pathogenic bacteria from the liquid phase during anaerobic digestion. Next, our study examined the impact of anaerobic digestion on virus adsorption to soil following land application of treated wastewater. Anaerobic digestion increased retention of viruses to the soil and decreased mobility of attached viruses through the soil matrix, as compared with untreated wastewater. Anaerobic digestion removed compound( s) that interfered with virus adsorption to soil. Initial characterization showed that these compound(s) were less than 100 kDa in size These compound(s) interfered with hydrophobic and electrostatic interactions used by the viruses for adsorption to soil. Futhermore, compound(s) less than 10 kDa in size caused inactivation ofMS2. Our results show the critical role of indigenous microflora and the retained biofilm during anaerobic digestion in a fixed-film system. The indigenous microflora and attachment to the biofilm causes decimation of indicator and pathogenic organisms from flushed dairy manure wastewater, thereby reducing the environmental load of these organisms during land application. Anaerobic digestion increases the retention of residual viruses within the soil matrix, therefore reducing the likelihood of contaminating groundwater.

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CHAPTER 1 INTRODUCTION Livestock are known reservoirs of pathogenic organisms and these organisms are shed with feces. Also, animal husbandry generates over 1.6 billion tons of manure per year in the United States (Altekruse et al., 1997). Pathogenic organisms in manure may be introduced to the environment by land application. Application of solid manure (as fertilizer) or diluted manure slurry (for fertirrigation) to land is a common management strategy used by concentrated animal feeding operations (CAFOs). Although this practice provides nutrients for crop production, pathogenic organisms may persist in manure, and potentially introduced to the environment and eventually to the herd and public (Hill, 2003). In regards to public health animal manure is a source of food contamination and has been linked to outbreaks of foodbome illnesses (Park and Diez-Gonzalez, 2003) Food can become contaminated with pathogenic organisms associated with manure during food processing (i e., slaughter) or by land application (i.e. contamination of uncooked fresh produce) {Troutt et al., 2001). Therefore, pathogenic organisms introduced by manure into the food chain can result in outbreaks of foodbome illnesses. Although viruses and protozoa may also be associated with foodbome illnesses, bacteria have been found to be the most common cause of outbreaks in the United States. The Centers for Disease Control and Prevention (CDC) reported 75% (655 of 878) of foodbome-disease outbreaks from 1993 to 1997 were of bact eria l origin (Centers for Disease Control and Prevention 2000). Furthermore, the Economic Research Service 1

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2 (ERS) of the United States Department of Agriculture estimated the annual cost associated with five bacterial agents of foodbome illnesses to be $6.9 billion. Effective treatment strategies must be used to reduce the levels of pathogenic bacteria in manure. However, to provide an incentive to implement new technology on CAFOs, treatment strategies should not only reduce the level of pathogenic organisms, but also offer on-farm and environmental benefits. Therefore, anaerobic digestion is a treatment strategy that can be used to reduce pathogen levels in manure and offer on-farm benefits such as bioenergy production, odor reduction, and biofertilizer production A fixed-film anaerobic system at the University of Florida Dairy Research Unit (DRU) has been shown to decrease pathogen levels and supply bioenergy and biofertilizer for on farm use (Davis et al., 2001 Wilkie, 2000) The persistence of pathogenic organisms in manure can potentially result in public health concerns. Current manure management strategies used by CAFOs may be improved to control the levels of pathogenic bacteria in manure Improvements should include implementing a manure treatment technology that offers on-farm and environmental benefits in addition to controlling pathogenic bacteria associated with manure. Thus, anaerobic digestion i s a viable option that offers pathogen control and on farm benefits. The fixed film anaerobic system used by the DRU has been shown to offer on-farm benefits while reducing the level of pathogens from manure. However the factor(s) that contribut e to p a thogen reduction during fixed-film anaerobic digestion and the effect on pathogen transport through soil following land application of treated wastewater remains unknown Therefore the purpose of the current study was to : 1) d e t e rmine th e factor(s) that contribute to pathogen reduction du ring fixed-film anaerobi c

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3 digestion and 2) determine the effect of fixed-film anaerobic digestion on virus transport through soil after land application.

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CHAPTER2 PATHOGENIC ORGANISMS IN BOVINE MANURE AND THEIR REDUCTION BY ANAEROBIC DIGESTION Pathogenic bacteria that pose herd and human health concerns are associated with manure (Bicudo and Goyal, 2003, Huston et al., 2002, Wang et al., 1996, Wells et al 1991) Furthermore, pathogenic bacteria can survive in manure for several days once voided from the animal (Himathongkham et al., 1999). Therefore, exposure to manure can potentially cause animal diseases, resulting in lost revenues for the animal operation Also, environmental contamination may result in human exposure to zoonotic organisms which may lead to potential diseases. Environmental contamination and transmission of zoonotic organisms to humans from livestock manure has been the focus of recent legislation to improve manure handling and treatment strategies (U.S. Environmental Protection Agency 2003). Another concern with manure is the emergence of antibiotic resistant strains of pathogenic bacteria. The emergence of antibiotic resistant pathogenic bacteria from manure of antibiotic-fed animals can pose severe herd and human health issues (Dargatz 1998) Therefore, the threat of antibiotic resistant pathogenic bacteria requires that better and more effective pathogen reducing manure management strategies be implemented by animal operations. Pathogenic Organisms in Dairy Cattle Manure Several types of pathogenic bacteria have been isolated from animal manure (Pell 1997): E. c oli O157 :H7 Salmon e lla spp., Liste r ia monocytog e n e s and Mycoba c t e r iu m paratuberculosis Patho ge nic viruses and proto z oan ha v e also been isolated from animal 4

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5 manure (Chang et al., 1997, Olson et al., 2004, Valle et al., 1999). Some" organisms are shed in high concentrations when the animal displays illness or may be shed in feces where the animal does not exhibit any clinical signs of disease. In either case, manure is a source of pathogenic organisms that may cause either human or herd diseases. Zoonotic Bacteria Escherichia coli 0157:H7. Escherichia coli 0157:H7 is an enterohemorrhagic strain of E.coli, which produces a Shiga-like toxin known as verocytotoxin. The verocytotoxin is a causative agent of several diseases such as, hemolytic uremic syndrome (HUS), thrombocytopenic purpura, and hemorrhagic colitis (Pell, 1997, Prescott et al., 1999, Wells et al., 1991). This organism is considered an emerging pathogen and has been isolated from raw beef and milk products (Callaway et al., 2003). Dairy cattle serve as a reservoir of E coli 0157:H7, where younger animals harbor the organism more so than mature animals. Wells et al. (1991) measured E. coli 0157:H7 concentrations in fecal samples from cattle in herds associated with two sporadic cases of hemolytic uremic syndrome and an outbreak of gastroenteritis. The investigation revealed that 2.3% of calves (5 of210), 3.0% of heifers (12 of394), and 0.15% of adult cows (1 of662) shed. coli 0157:H7 in feces. Doyle et al. (1997) also found that E. coli 0157:H7 is isolated predominantly from young animals, with the highest rate of isolation from postweaned calves. Zhao et al. (1995) found that E. coli 0157:H7 was shed more frequently by weaned calves (4.9 to 5.3%) than preweaned calves (1.5 to 2.9%). These results show that not only are dairy cattle reservoirs of E. coli 0157:H7, but younger animals tend to harbor the organism more so than adult animals.

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6 Escherichia coli O157:H7 can not only be shed in feces, but also proliferate and persist in feces once introduced into the environment. Wang et al. (1996) showed that. coli O157:H7 could survive and proliferate in bovine feces up to 21 days at 37C, 49 to 56 days at 22C, and 63 to 70 days at 5C, although no growth occurred at 5C. Nicholson et al. (2005) found that E.coli O157:H7 could survive for 93 days in dairy manure slurry stored at less than 20C. However, survival decreased to 32 days following land application without storage. Park and Diez-Gonzalez (2003) found that E. coli O157:H7 could survive in cattle manure for at least 25 days at room temperature. Salmonella spp. Salmonella spp. is the most common pathogen associated with livestock manure (Stehman, et al., 1996). These organisms are facultative anaerobic, gram-negative rods. These organisms are non-species specific ( e.g., equine, swine, poultry, and bovine), can be transferred from animals to humans through undercooked meat or raw eggs, and are causative agents of typhoid fever, enteric fevers, septicemia, and gastroenteritis. Salmonella spp. causes gastroenteritis in humans and can be fatal to young, elderly, or immuno-compromised individuals (Dargatz, 1998). From 1993 to 1997, 55% of bacterial foodbome-disease outbreaks in the United States were caused by Salmonella spp (Centers for Disease Control and Prevention, 2000). The Economic Research Service (ERS) estimated annual economic loss due to foodbome Salmonella infections in 1996 and 2003 was $3.5 and $2.9 billion, respectively (Buzby et al., 1996 ERS, 2003, Frenzen et al., 1999). The emergence of antimicrobial resistant Salmonella spp. has become a major health concern. In particular, S. typhimurium DT104 has d e veloped resistance to various antibiotics includein g ampicillin, sulfonamides, tetracycline chloramphenicol and

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7 streptomycin (Dargatz, 1998). Park and Diez-Gonzalez (2003) found that S. typhimurium DT104 could survive in cattle manure for at least 25 days at room temperature. During slaughter, meat can potentially become contaminated with S. typhimurium DTl 04. Galland et al. (2001) identified 59 isolates as S. typhimurium in a survey of five slaughter establishments processing cull (market) dairy cattle. Out of the 59 isolates, 35 were obtained from one facility, where 88.6% of those isolates were confirmed as S. typhimurium DT104. Salmonella spp. are commonly associated with dairy cattle. Huston et al. (2002) found that out of 2,283 individual fecal samples and 15 bulk milk tank samples from five dairy herds, 11 % of calves, 56% of mature cows and 20% of bulk tank samples were positive for Salmonella spp. The study also found that mature cows were 21 times more likely to be shedding Salmonella spp. than were unweaned calves. Furthemore, no clinical signs of salmonellosis were identified in any of the adult animals in the five herds The study concluded that Salmonella spp. can be shed in feces of mature healthy cows, however bacterial concentrations were not determined during the study. Dairy cattle can also serve as a vehicle for Salmonella spp. contamination of meat. Troutt et al. (2001) measured prevalence of Salmonella spp. in five beef slaughter establishments in five different regions of the United States. The investigators found a 23 .1 % overall prevalence of Salmon e lla spp. Additionally, the highest prevalence (54.5%) and th e lowest (4.3%) prevalence were found during the summer sampling period and in two different locations. The investi g ators were able to detect Salmonella spp. in all th e e stablishments survey e d These r e sults suggest that the prevalence of Salmon e lla spp. is not dependant on seasonal variations nor is the organism isolated to

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8 one region of the country. Galland et al. (2001) determined Salmonella serotype diversity and prevalence from the same samples obtained in the study by Troutt et al. (2001). The investigators identified 58 serotypes of Salmonella, where Salmonella ser. Montevideo was the most frequent (21 %). Other serotypes (Muenster, Kentucky, Mbandak:a, Senftenberg, and Typhimurium) were found in all establishments surveyed. Campylobacter spp. Campylobacter spp. are microaerophilic, gram-negative vibriod cells. They are usually found in the oral cavity, reproductive organs, and intestinal tract of animals and humans. Two species of particular concern are C. jejuni and C. coli. These organisms cause human enteritis and are associated with livestock. Campylobacter spp. may enter the supply of raw milk or water from bovine feces (Wesley et al., 2000) Healthy cattle are potential reservoirs of C. jejuni and C. coli and the incidence of disease is seasonal (Wesley et al., 2000). Mycobacterium paratuberculosis. Mycobacterium paratuberculosis is an aerobic, acid-fast, rod-shaped organism. This slow-growing, fastidious organism generally requires the presence of mycobactin, a growth factor that serves as an iron chelator, to proliferate (Grant et al., 1996). Mycobacterium paratuberculosis is the causative agent for Paratuberculosis (PTB), or Johne's disease, in cattle and has been implicated in Crohn's disease in humans (Grant et al., 1996). Paratuberculosis is a chronic, granulomatous intestinal disease that is manifested by nonresponsive diarrhea, progressive weight loss, and death. Mycobact e rium paratuberculosis infections are not confined to specific tissues and the organism can be isolated from several organs (Hines et al., 1987) This organism has also been isolated from uterine flush fluids of infected dairy cattle (Rohde et al., 1990).

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9 Several techniques have been developed to detect M paratuberculosis. Collins et al. (1990) described the use of radiometry for detection. A study of nine infected dairy herds in Wisconsin showed that radiometric detection using filter-concentrated manure samples detected 92% of M paratuberculosis from feces, while conventional culture techniques using HEY agar only detected 60%. Molecular detection of M paratuberculosis has also been studied. Millar et al. (1996) found that IS900, an unusual DNA insertion element unique to M paratuberculosis, can be used to detect this organism in pasteurized milk. Paratuberculosis has an adverse effect on milk production (Hernandez and Baca, 1998). Mycobacterium paratuberculosis can be shed in milk in high concentrations (Sweeney et al., 1992) and can survive pasteurization temperatures (Grant et al., 1996). This organism can also be transmitted in utero (Seitz et al., 1989) and by herd-to-herd contact (Collins et al., 1994). The prevalence of this organism at a dairy can be attributed to environmental conditions, manure-handling practices, newborn calf care, and grower calf care (Goodger et al., 1996). Listeria monocytogenes. List er ia monocytogenes are facultative anaerobic, gram positive, rod shaped organisms. These organisms cause listeriosis in animals and humans (George et al. 1996, Jensen et al. 1996, Mussa, et al. 1999). Jensen et al. (1996) found that L. monocytogenes isolated from humans and cows were of the same serotype. This implies that milk contaminated with this organism can possibly infect humans. The ability of this organism to survive in food causes concern for human health (George et al., 1996). Gohil et al. (1996) found thatL. monocytog enes can survive up to three days in labneh and houmos two traditional Arabic foods. Listeria monocytogenes

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10 have also been shown to survive in acidic foods ( e.g., yogurt, cottage, mozzarella, and cheddar cheeses) by induction of the acid tolerance response (ATR) (Gahan et al., 1996). The ATR mechanism involves the synthesis of proteins, including outer membrane and heat shock proteins, which provide a mechanism for maintaining intracellular homeostasis in suboptimal, or lethal, environments. Another concern for human health is that antibiotic resistant strains of these organisms have been detected. Erythromycin resistant (EmJ and tetracycline resistant (TetJ Listeria spp. has been isolated from food (Roberts et al., 1996). The use of high pressure (600 MPa) has been studied as a possible treatment process for food. Mussa et al. (1999) found that the use of high pressure in the pasteurization reduced the concentration of L. monocytogenes in milk. Mastitic Bacteria Staphylococcus aureus. Staphylococcus aureus are gram-positive, facultative anaerobic cocci. These organisms are common inhabitants of the skin and mucous membranes of warm-blooded animals. Staphylococcus aureus are causative agents of peracute, acute, chronic, subclinical mastitis in dairy cattle and can be excreted in milk (Umeki et al., 1993). Subclinical infections are the most common form of mastitis caused by S. aureus, however this organism can also cause clinical mastitis (Elbers et al., 1998). Staphylococcus aureus induced mastitis (i.e., contagious mastitis) is characterized by invasion of the mammary gland tissues, where the mammary gland parenchyma is replaced by granulation tissue, fibrosis occurs, and multiple abscesses form (Cullor, 1993). Although antibiotic therapy has been used to treat infections, resistant strains of S. aureus associated with mastitis have emerged in recent years (Malinowski and Klossowska, 2003).

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11 Streptococcus spp. Streptococci are facultatively anaerobic gram-positive cocci. These organisms have a fermentative metabolism where lactate is the primary product. These organisms are common inhabitants of the mouth and upper respiratory tract of vertebrates. Several species of streptococci are causative agents of bovine mastitis. A streptococcus responsible for contagious mastitis is S. agalactiae. Streptococcus uberis and S. dysgalactia are associated with environmental mastitis. Contagious mastitis infections caused by S. agalactiae (Lancefield group B) can be either clinical or subclinical. Infections involve adherence of the organism to mammary gland cells. Although, Lammers et al. (2001) showed poor adhesion to mammary gland cells by S. agalactiae, fibrinogen-binding proteins secreted by the organism have been shown to aid in adhesion to mammary gland cells (Jacobsson, 2003). A single strain of S. agalactiae has been shown to be the causative agent of mastitis. Merl et al. (2003) studied 79 streptococcal isolates from subclinical mastitis of 54 cows. The study found that a single strain of S agalactiae were responsible for all the infections. In addition to mastitis, S agalactiae can cause severe invasive disease in humans, especially in neonates (Bohnsack et al., 2004). Evidence suggests that S. agalactiae may be a zoonose, being transmitted from cows to humans and visa versa, although the risk of transmission between species is considered low (Sukhnanand et al., 2005). Environmental mastitis infections may be related to environmental factors such as insect population (Yeoman and Warren 1984) and bedding materials (Hogan et al., 1990). "Summer mastitis is a teat infection caused by several organisms including S. dysgalactiae and is spread by a fly, Hydrotaea irritans (Yeoman and Warren, 1984).

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12 Summer mastitis infections caused by S. dysgalactiae were found to be five times more prevalent in herds without fly control versus herds with fly control (Nickerson et al., 1995). The type of material used for bedding can result in increased herd exposure to environmental mastitic streptococci. Care and type of bedding material (i.e., organic or inorganic) used in freestall barns have a direct impact on exposure to environmental mastitic streptococci (Bey et al., 2002). Hogan et al. (1990) found that environmental mastitic streptococci were able to proliferate in organic bedding material and increase the concentration of these organisms on teats. Zehner et al. (1986) found S. uberis was able to proliferate in various types of organic bedding ( e.g., recycled manure, chopped straw, hardwood chips, paper, and softwood sawdust) Additionally, Todhunter et al. (1995) reported streptococci concentrations of 6 to 7 log10 CFU/g dry weight of various bedding materials (i.e., pelleted com cobs, recycled manure, and wood shavings) throughout the year. Although streptococci concentrations did not vary with seasonal conditions, the rate of environmental streptococcal mastitis (reported as intramammary infections per cow per day) was highest during the summer and lowest during the winter. These results suggest that the use of organic bedding material can lead to environmental mastitis by providing conditions for mastitic streptococci to survive and proliferate. Pathogenic Viruses BVDV. Bovine virus diarrhea is an infectious disease in cattle caused by a retrovirus, BVDV. BVDV is a member of the Pestivirus genus, within the Flaviviridae family. The attachment and entry of flavivirus is mediated by the envelope (E) protein ( 5 0 kDa), a major glycoprotein on the virus particle (Lee and Lobigs, 2000). BVDV is found in oculonasal discharges and is transmitted by close and direct contact within the

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13 herd. An important feature of this disease is that an infected pregnant female can give birth to a persistently infected calf that will excrete BVDV throughout its life (Valle et al., 1999). Valle et al. (2000) studied BVDV sero-conversion (a surrogate measure for incidence) in Norwegian dairy herds and found that there was a steady decline in sero conversion risk. Although sero-conversion continued over time, the decrease was attributed to effective control of major risk factors. These factors include the use of common pastures, purchasing animals associated with BVDV, and herd-to-herd contact over pasture fences (Valle et al., 1999). Bovine rotavirus. Bovine rotavirus is a member of the Reoviridae family, has a genome that consists of 11 segments of dsRNA, and is enclosed by a triple-layered capsid Rotaviruses are a common cause of diarrhea in calves and humans (Chang et al., 1997, Klingenberg et al., 1999). There are three groups ofrotaviruses: A, B, and C. Group A is a common cause of neonatal calf enteritis (Klingenberg et al., 1999). Group B has been implicated in sporadic cases of diarrhea in calves (Chang et al., 1997) Group C has been suggested as a common enteric pathogen in animals and humans, but are difficult to cultivate because they are fastidious in their in vitro cell culture requirements (Alfieri et al., 1999). Activation of rotavirus infectivity requires the presence of exogenous proteases, which cleave the VP4 protein. Replication is completely cytoplasmic, where rotaviruses supply necessary enzymes to replicate dsRNA. Subviral particles form and maturation occurs through budding of the endoplasmic reticulum (ER) Finally, viral particles are released through cell lysis.

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14 Two major proteins, VP4 and VP7, on the outer shell ofrotaviruses have been studied extensively (Alfieri et al., 1999, Chang et al., 1997, Klingenberg et al., 1999). The genes of these two outer capsid proteins are used for molecular detection of rotaviruses using reverse transcription polymerase chain reaction (RT-PCR). Buesa et al. (1996) found that RT-PCR is highly effective for G (VP7 gene) and P (VP4 gene) genotyping of rotaviruses, but not substantially more sensitive than enzyme linked immunosorbant assay (ELISA) and electron microscopy (EM) for detection in fecal and environmental samples. To control the spread of this pathogen preventive measures should be aimed at better hygiene herd management, and vaccination programs (Klingenberg et al. 1999). Parasitic Protozoa Cryptosporidia. Cryptosporidia are protozoan parasites that are commonly associated with human and livestock manure (Olson et al. 2004) The organism ex i sts as an oocyst during the infectious stage. Oocyst are biologically dormant easily transported in water and can survive drinking water treatment (i.e., chlorination). Although there are several species of cyptosporidia (e.g., C. andersoni and C. muris) only C. parvum is clinically significant. Cryptosporidium parvum is the causativ e ag e nt of cryptosporidiosis in humans, which is characterized by severe diarrhea and dehydration. Cattle are consider e d the major reservoir of C. p a rvum (Olson et al., 2004). Younger calves are more susceptible to infection and are more likely to shed oocysts during infection than ar e older animals Sischo et al. ( 2 000) measured the prevalence of cryptosporidia in dairy cattle and cal v es. The invest ig ators detected Cryp tosporidium in 10 of 11 farms surv e y e d F urthermore calv e s (0 to 3 w ee ks old) had th e highest prevalence of cryptosporidia infections among older cal ve s (::: 4 weeks old) and mature

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15 cows. Additionally, the investigators evaluated several risk factors for spreading crytosporidia among calves. Contact between calves and frequent bedding changes(> 12 times per year) were found to be the two main management risk factors associated with fecal shedding of cryptosporida. Younger calves are more likely to become infected and shed oocysts. However, adult animals are can shed oocysts where infections are asymptomatic (Graczyk et al., 2000). The field spreading of contaminated calf manure can potentially introduce oocysts to the watersheds (Walker and Stedinger 1999). To contaminate watersheds, oocysts must transported by manure and released into a watershed. Schijven et al. (2004) measured oocyst release from manure by water application either as a mist or droplets. Calf and cow manure were used separately or mixed together in the study to simulate manure management practices at the dairy operation. The study found that oocysts were more readily released from calf manure than cow manure. The larger coarse particles (i.e., undigested hay or grain) in cow manure do not readily partition into the aqueous phase. However, calf manure consisted of finer colloidal particles, which partitioned easily into the aqueous phase. The differences in manure composition are directly impacted by the difference in diet among cows and calves. The study also found that droplet size and water application rate was directly proportional to oocyst release rates. This result suggests that during heavy rainfall events, oocysts can be released from manure and potentially be transported in runoff. Anaerobic Digestion and Pathogenic Organisms Anaerobic Digestion Anaerobic digestion is the natural process of converting complex organic compounds to methane and carbon dioxide by a series of symbiotic microbial interactions

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16 in the absence of oxygen. The process of anaerobic digestion is utilized as a means of degrading organic matter in the environment, providing nutrition for ruminants, and treating different industrial and agricultural organic waste products. Ruminant organisms have evolved to utilize anaerobic digestion for degrading and deriving energy from otherwise indigestible plant material. Anaerobic degradation under controlled conditions provides an effective method for treating organic wastes. The development of enclosed systems (i.e., anaerobic digesters) has enhanced the process of anaerobic digestion by providing an optimum environment for methanogenesis. Anaerobic Digesters Anaerobic digesters are bioreactors designed to optimize the process of anaerobic degradation by providing an environment under controlled conditions to facilitate the growth of the anaerobic bacteria. These systems are designed for wastewater stabilization and energy production (i.e., methanogenesis). Other benefits derived from anaerobic digesters are odor reduction (Powers, 1999) and bacterial pathogen decimation (Kearney, 1993). Several types of digesters have been studied for treating manure. These include anaerobic lagoons, continuous stirred tank reactors, plug-flow digesters, and fixed-bed reactors. Anaerobic Lagoons. Preceding digesters, anaerobic lagoons have been used to treat animal manures. However, lagoons do not offer biogas recovery for energy production. Development of covers over lagoon systems permitted biogas recovery for energy use. These systems are operated at ambient temperature, resulting in seasonal changes in biogas production. Covered anaerobic lagoons require hydraulic retention times (HRTs) in excess of 40 days. These systems have been found to achieve COD reductions of70% to 94% and produce biogas with various methane contents (70% to

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17 88% C"4) (Cheng et al., 1999, Safley and Westerman, 1992, Williams and Frederick, 2001). Continuous Stirred Tank Reactors. Continuous stirred tank reactors (CSTRs) are insulated, cylindrical tanks that are either continuously or intermittently fed, where the volume of effluent equals the volume of influent. These systems are used for methane recovery from livestock manure that usually contains a 5% to 12% total solids concentration. The contents of CSTRs are mixed either by mechanical methods or recycling biogas through the liquid. These systems are operated at HRTs of 10 to 20 days (Wilkie and Colleran, 1989) and maintained at either mesophilic (-35C) or thermophilic (-55C) temperatures, although psychrophilic (5C to 20C) operating temperatures have been studied (Zeeman et al., 1988). Plug-flow Digesters. Plug-flow digesters are systems used to treat scraped manure with an 11 % to 14% total solids concentration (Hills 1983, Hills and Mehlschau, 1984 Liu, 1998). They are usually long rectangular tanks often built into the ground and equipped with an impermeable plastic cover for biogas recovery. Manure is not mixed as it moves through the system as a combined mass or plug". Plug flow systems usually operate at HRTs in excess of 30 days. Fixed-bed Reacton. Fixed-bed anaerobic reactors use a support medium to reta i n anaerobic organisms A fixed-bed system is constructed with an internal support media to provide surface area for the attachment and retention of anaerobic bacteria as a biofilm. This advancement allows for the retention of anaerobic bacteria within the di g ester independ e nt of HRT (Wilkie 2000) Adh e sion of anaerobic bacteria to inert support media, facilitating biofilm formation is influenced by bacterial surface properties

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18 and support media characteristics (Verrier et al., 1987). The surface texture and porosity of the support media have a significant impact on system performance, where open-por~ surface texture and high porosity results in higher biofilm efficiency (Show, 1999). A major advantage of fixed-bed technology is the immobilization of methanogens, which grow at a slow rate (Wilkie and Colleran, 1989). Fixed-bed technology allows for the bacteria to be retained within the digester, resulting in a decrease in HRT needed to digest wastewater (Wilkie, 2000). This type of wastewater management system is capable of treating larger volumes of dilute wastewater per unit time than conventional systems (Wilkie, 2000). Fixed-bed anaerobic reactors are suited for treating slurry with low ( < 1 %) total solids concentrations. Microbial Diversity in Anaerobic Digesters The microbial ecosystem within anaerobic digesters is primarily a consortium of bacteria that are involved in synergistic interactions for the digestion of organic compounds for biogas production (Bitton 1994, Wilkie and Colleran, 1987). The process of digestion involves four groups of bacteria: 1) hydrolytic bacteria, 2) acidogenic bacteria 3) acetogenic bacteria, and 4) m ethanogenic bacteria. The four groups of bacteria are dependent on each other for maintaining an optimum environment for anaerobic digestion. Hydrolytic Bacteria Hydrolytic bacteria degrade complex organic material (e g polysaccharides proteins, lipids), yielding monomers that are subsequently utili z ed by the acidogenic bacteria Hydrolytic species inhabiting di g esters feed ruminant manure originate from org anisms that survive passage from the rumen and into feces. Vel az qu ez et al. (2004) identifi e d a novel species of Pa e n i ba c illus P favisporus sp. nov. This

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19 organism was isolated from bovine feces and found to produce a variety ofhydrolyic enzymes, including xylanases, cellulases, and amylases. Acidogenic Bacteria. The acidogenic bacteria convert the monomers ( e.g., monosaccharides, fatty acids, amino acids) to organic acids, alcohols, and ketones. Acidogenic bacteria include Propionium spp. that produces propionate and Butyrvibirio spp. Butyrvibirio fibrisolvens are gram-negative, non-sporeforming, curved rods. These organisms mainly utilize monoand disaccharides as substrates, resulting in the production ofbutyric acid (Bryant and Small, 1956) Acetogenic Bacteria. The acetogenic bacteria primarily utilize propionate and butyrate as substrates within anaerobic digesters. Syntrobacter wolinii utilizes propionate and Syntrophomonas wolfei utilizes butyrate, where both organisms form acetate, CO2 and H2 (Wilkie and Colleran, 1987) Metbanogenic Bacteria. There are two primary groups of methanogenic bacteria active in anaerobic digesters: 1) hydrogen-utilizing methanogens and 2) acetate-utilizing methanogens. Hydrogenotrophic methanogens utilize CO2 and H2 for methane production. These organisms are not primary methane producers in the digesters, however their activity maintains a low partial pressure of hydrogen to drive interspecies hydrogen transfer within digesters. Acetotrophic methanogens ( e.g., M e thanosarcina barkeri and Methanothrix soehngenii) utilize acetate to form methane (CH4). Methanosarcina bark e ri is usually found in systems operating at low HRTs whereas Methanothri x so e hngenii is found in systems operating at high HRTs (Wilkie and Colleran 198 7 ) Acetate is the primary source of m e thane and accounts for

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20 approximately 70% of the methane produced within digesters (Wilkie and Colleran, 1987). Pathogen Decimation during Anaerobic Digestion of Animal Manure Anaerobic digestion for stabilizing manure offers a non-chemical solution to control pathogenic organisms. Anaerobic digesters provide a controlled and optimum environment for the natural process of anaerobic degradation. The use of anaerobic technology to control pathogenic organisms has been studied for several systems. The survival of pathogenic organisms during anaerobic digestion of animal manure is influenced by several factors. Operating temperature and HRT have been shown to influence the rate of pathogen decimation during anaerobic digestion However, environmental conditions during anaerobic digestion may also contribute to the rate of pathogen decimation. The pH of digester contents affects pathogen survival during digestion and systems have been designed to use this mechanism to reduce pathogen concentrations (Huyard et al., 2000). Substrate availability, microbial competition for substrates, and bacterial production of antibacterial compounds may also contribute to decimation of pathogenic organisms during anaerobic digestion (Kearney et al., 1993, Sahlstrom, 2003, Smith et al., 2005). Temperature. One of the most important parameters influencing the rate of reduction of pathogenic organisms during anaerobic digestion is operating temperature. Temperature influences the kinetics of anaerobic digestion, which affects environmental conditions of digester contents. Anaerobic digesters are usually operated at either mesophilic ( 35C) or thermophilic ( 55C) temperatures. Mesophilic operation maintains the digester at or near the temperature pathogens are exposed to in the animal. However, anaerobic digestion at this temperature range has

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21 been shown to achieve significant reductions of pathogenic organisms. Duarte et al. (1992) demonstrated that mesophilic anaerobic digestion with a 15-day HRT resulted in a 3-log10 reduction of total coliforms, fecal coliforms, and streptococci from swine manure. Thermophilic operations maintain digester temperature in excess of that found within the animal. The higher operating temperatures have been shown to produce significant reductions of pathogens in shorter amounts of time as compared to mesophilic digestion (De Leon and Jenkins, 2002). Decimation times of pathogenic organisms are in the range of a few hours during thermophilic digestion, whereas mesophilic operations require several days (Berg and Berman, 1980, Burtscher et al., 1998, Watanabe et al., 1997). Chauret et al. (1999) found that mesophilic digestion of sewage sludge achieved a 0.3 log1oreduction of cyptosporida oocysts after 20 days. However, Kato et al. (2003) found thermophilic digestion of biosolids resulted in> 2 log10 reduction of cryptosporidia oocysts after 1 h. Although thermophilic operation yields higher pathogen reductions, process stability has been cited as a disadvantage (Kim et al., 2002). Thermophilic operation results in rapid volatile acid production from fast-growing thermotolerant acidogens The rapid production of volatile fatty acids lowers the pH of the medium, inhibiting the growth of methanogens which grow at a slower rate. The low pH encountered in thermophilic operations reduces concentrations of pathogenic organisms. Hydraulic Retention Time. The HRT of a digester greatly influences pathogen survival during anaerobic digestion. The HRT is the average amount o f time a desired substrat e r e mains in the system and is a function of system capacity and feed rate. Th e longe r th e HRT the longer substrate is r e tain e d in the system, which lowers the amount

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22 of substrate that can be treated per unit of time. Systems that operate at long HRTs ~10 days) can achieve over 2 log10 reductions of pathogenic organisms from animal manure (Duarte et al., 1992). Pathogen survival is influenced by HRT, where retention in a suboptimum environment results in decimation. During the process of anaerobic digestion, environmental conditions become stressed due to lack of available nutrients and pathogens cannot maintain a high population density as compared to concentrations inside the animal. Influence of Temperature and HRT. The combination of operating temperature and hydraulic retention time are regarded as the main factors impacting pathogen survival during anaerobic digestion. Mesophilic operations with long hydraulic retention times and thermophilic operations with short hydraulic retention times can reduce pathogen levels from animal manure. However, pathogen reduction during anaerobic digestion is not based solely on operating temperature and hydraulic retention time (Hill 2003). Operating temperature and hydraulic retention time may influence the alteration of the physico-chemical properties of wastewater during digestion. Alteration of the physico chemical properties may have direct impacts on pathogen acclimation and survival in an anaerobic digester environment. Volatile Fatty Acids. Short-chain volatile fatty acids (e.g., acetate propionate and isobutyrate) produced by_ acidogens during anaerobic digestion can contribute to pathogen decimation. Abdul and Lloyd (1985) demonstrated growth inhibition o f E. coli by various short-chain fatty acids. Cells were grown in 0.1 % glucose under anaerobic conditions. Short chain volatile fatty a cids were added to the medium a s potassium salts during early log phase The authors d e monstrated inhibition of growth by acetate (60 and

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23 120 mM), propionate (52 and 104 mM), and isobutyrate (60, 90, and 180 mM). Kunte et al. (1998) demonstrated inactivation of S. typhi in cattle manure by volatile fatty acids during anaerobic digestion for 30 days ( control) and 15 days ( experimental). The feed for the experimental digester was supplemented with 1 % glucose daily. The decimation times (T90), the time required to achieve a 1 log10 reduction, were significantly lower for experimental digesters producing high volatile fatty acid concentrations (5 g/L} as compared to control digesters producing lower volatile fatty acid concentrations (0.1 g/L) However the authors did not report operating temperature of each digester. Kwon and Ricke (1999) demonstrated growth inhibition of Salmonella typhimurium by propionic acid and sodium propionate Cells were grown in tryptic soy broth (TSB) with various concentrations of propionic acid or sodium propionate. The culture medium was adjusted to pH 7.0. Inhibition of S typhimurium was observed with propionic acid and sodium propionate concentration ::: 25 mM. Effect of pH. The pH of digester contents has significant effects on pathogen survival during anaerobic digestion. The production of short-chain volatile fatty acids during anaerobic digestion lowers the pH of the medium (Munch et al. 1998) Short chain volatile fatty acids can contribute to pathogen decimation by lowering environmental pH. However during anaerobic digestion short-chain volatile fatty acids serve as substrates for subsequent methanogenesis. In a single-phase digester, where acid i ogenesis and methanogenesis occur in the same reactor, the proce ss of methanogenesis counteracts the pH effe cts of volatile fatty acids with the production of bicarbonat e and the consumption o f a c e tat e Bicarbona t e buffers the di g ester contents maintaining a pH o f 6.5 to 7.0. T hus pH may not contribute to pathogen decimation

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24 during single-phase anaerobic digestion. Two-phase anaerobic digesters have been developed that separate the acidogenic phase from the methanogenic phase of anaerobic digestion. The acidogenic phase is usually operated at theromophilic (2:55C) temperatures (Yu and Fang, 2000) and short HRTs (Huyard et al., 2000), resulting in rapid growth of thermotolerant acidogenic organisms The effluent from the acidogenic phase is fed to the methanogenic phase, which is operated at mesophilic (-35C) and long HRTs (2: 10 days) to accommodate the slow growth rate ofmethanogens (Huyard et al., 2000). During the methanogenic phase, short-chain volatile fatty acids are converted to acetate, hydrogen, and carbon dioxide. These products serve as substrates for acetotrophic and hydrogenotrophic methanogens. Acidogenic and methanogenic phases can be operated and controlled independently, allowing for optimization of each phase without adverse affects to methanogenic and acidogenic populations (Ince, 1998). Phase separation allows for pathogen reduction during the acidogenic process due to lower pH resulting from the production of short-chain volatile fatty acids. Solids Concentration. The survival of pathogens during anaerobic digestion of manure is influenced by the amount of solids present in digester contents. Pathogens can become readily associated with organic particl e s in the e nvironment (Grossart et al., 2003) Nutrients absorb onto the surfaces of organic particles where pathogens can adhere and use the solids as a food source (Goulder 1977) Bacterial adherence may be achieved by the use of fimbria e (Galfi e t al. 1998) S layers (Kotiranta e t al. 1998), and pili (Orndorff et al 2004). London-van der Waals forces hydrophobic, and electrostatic int e ract i ons are the main mechanism s for bacterial and viral adh e sion to solids (Ash 1 979 Dani e ls, 1980) In addition e xopolym e ric substanc e s and cell surface proteins also

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25 contribute to adhesion to particles (Wicken, 1985). During anaerobic digestion, pathogens can attach to organic particles, supplying the organism with a nutrient source, increasing the likelihood of survival. Kumar et al. (1999) found decreased pathogen reductions from cattle dung using anaerobic digestion at mesophilic temperature with increased loading of volatile suspended solids concentrations Substrate Availability. The availability of substrate, as organic carbon, has a significant role in pathogen survival during anaerobic digestion. The amount of organic carbon available for biological degradation and assimilation is represented by biochemical oxygen demand (BOD). Anaerobic digesters can significantly reduce BOD concentrations in animal manure (Chin and Ong, 1993). The reduction in BOD creates a substrate-limiting environment, and pathogens are not readily suited to such stressed conditions. Pathogens associated with manure are voided from the intestines of the animal, an optimum environment. Once voided from the animal, pathogens in manure are exposed to environmental conditions. As manure enters an anaerobic digester, the amount of available substrate may fluctuate and pathogens have to be capable of adapting to changes in substrate fluctuations. The organic material that is available for biological degradation is mainly used for the formation of methane. During optimized anaerobic digestion there is little available substrate to maintain pathogen concentrations found in the animal (Kearney et al., 1994). Microbial Competition. Anaerobic digesters are optimum environments for methanogenesis and are operated for supporting the proliferation of organisms involved with the process of anaerobic d iges tion. Therefore, organisms involved with carbon flow through anaerobic digestion readily degrade and assimilate available organic carbon that

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26 is fed to the system. Affinity for necessary substrates to support proliferation is higher for organisms involved in the process of anaerobic digestion than for pathogenic organisms. Saturation constants (Ks) for anaerobic digestion bacteria and substrates are lower than for other bacteria (Mosche et al., 1998). The lower Ks values result in a higher affmity for substrates for bacteria involved with anaerobic digestion (Song et al., 2004). Results from the aforementioned studies suggest that pathogens cannot readily utilize the limited amount of substrate to support a high population as compared with bacteria associated with anaerobic digestion, resulting in pathogen decimation. In addition to higher affinity for substrates, differences in concentrations of digester microflora and pathogens influences microbial competition wi~hin anaerobic digesters Pathogens are usually found at lower concentrations than nonpathogenic bacteria, unless voided in high numbers by an infected animal (Huston et al., 2002). Anaerobic digesters are predominated by bacteria involved with anaerobic digestion and are present in high concentrations (107 to 108 CFU/ml) (Solera et al., 2001). Pathogens are usually loaded to the system in lower concentrations as compared to anaerobic digestion bacteria already present in the system. The difference in population density and the higher affinity for substrates give anaerobic digestion bacteria a survival advantage in anaerobic digesters, leaving little opportunity for bacterial indicators and pathogens to become established. Antimicrobial Products of Bacteria. Bacteria are in constant competition with other bacterial species for available nutrients in natural environments. Different genera of bacteria produce substances that have antibacterial properties The type of substances produced and the resultin g effe cts can be specific for different sp e ci e s within the same genus Bacteriocins and colicins are antimicrobial compound s that are produced by

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27 bacteria to give the organism a competitive advantage in natural environments. The role ofbacteriocins and colicins has not been extensively studied in regards to pathogen reduction during anaerobic digestion. However, organisms that can be found in animal manure and subsequently in anaerobic digesters have been shown to produce bacteriocins and colicins. Holo et al. (2002) reviewed bacteriocins from propionate producing bacteria that were effective inhibitors of gram positive and gram negative organisms. Lyon and Olson (1997) isolated colicins produced by Escherichia coli ECL12 (isolated from bovine feces) that inhibited the growth of pathogenic E.coli strains Bacteriocins and colicins may be present and active in digesters, contributing to pathogen decimation during anaerobic digestion. Summary Pathogenic organisms present in animal manure pose a serious herd and human health concern. Manure management practices have become the focus of recent legislation to control the transmission of zoonotic organisms to the environment. Anaerobic digestion has been shown to reduce the level of pathogens from animal manure. The rate of pathog e n decimation during anaerobic digestion has been related to operating temperatures and hydraulic retention times. However other factors, such as pH, volatile fatty acid concentrations, microbial competition and substrate affinity have a significant role in the fate of pathogenic organisms during anaerobic digestion. Although systems designed for the purpose of pathogen reduction focus on operating temperature, reactor configurations that can optimi z e other factors that contribute to pathogen reduction may pro v e effici e nt and superior to current s ystems for manure wastewat e r sanitization.

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28 Purpose of Study The purpose of the current work was to identify the factor( s) that contribute to the reduction of indicator and pathogenic bacteria during anaerobic digestion in a fixed-film system. Also, we sought to study the transport of residual viruses through soil following land application of anaerobically treated flushed dairy manure wastewater.

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CHAPTER3 INDICATOR AND PATHOGENIC BACTERIA REDUCTION BY ANAEROBIC DIGESTION: THE ROLE OF MICROBIAL COMPETITION AND SUBSTRATE LIMITATIONS Introduction Several pathogenic bacteria are known to be associated with dairy manure These include organisms that mainly infect dairy cattle, such as S. aureus and streptococci, and zoonotic organims, such as E. coli O157:H7 and Salmonella spp., which can cause infections in humans. Therefore, disposal of animal manure by land application raises environmental and public health concerns. Effective treatment of manure by anaerobic digestion can be achieved using well-managed systems. Anaerobic digesters have been shown to achieve 2 to 3 log10 reductions of a variety of indicator and pathogenic organisms from various substrates (DeLeon and Jenkins, 2002, Duarte et al., 1992). Decimation of pathogenic bacteria during anaerobic digestion may due to inhibitory effects caused by microbial interactions and competition for limited substrates. Inhibitory effects of indigenous micro flora may involve the production of secondary metabolites. Such metabolites are produced during stationary phase or idiophase and usually have antimicrobial properties (Riviere et al., 1975). Secondary metabolites include antibiotics, colicins and bacteriocins. These compounds are usually effective against strains related to the producing organism Lyon and Olson (1997) studied a bacteriocin designated colicin ECL 12 from E. coli isolated from swine manure Colicin ECL 1 2 was found to inhibit two strains of E. coli and 17 strains of E coli O157:H7. Howev e r there were no inhibitory effects found against selected gram 29

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30 positive pathogenic bacteria (e.g. S. aureus and L. monocytogenes). Bacteriocins may also demonstrate activity against both gram-positive and gram-negative organisms. Hyronimus et al. (1998) found that a strain of Bacillus coagulans, isolated from bovine manure, produced coagulin. Coagulin is a bacteriocin capable of inhibiting a variety of bacteria including strains of Bacillus spp., Enterococcus spp. and Listeria spp. Propionic bacteria have also been shown to produce bacteriocins. Holo et al. (2002) identified several bacteriocins produced from propionate producing bacteria. Volatile fatty acids (VF As) produced by bacteria have also been shown to display bacteriostatic or bacteriocidal effects Kunte et al. (1998) measured the effects of VF A concentrations on the decimal reduction time {T 90) of Salmonella typhi in anaerobic digesters. The authors used a KVIC model floating dome digester (9.5 L) operated at a 15-day retention time and fed cattle dung slurry (6% total solids). The investigators found that at a constant total VF A concentration ( 5000 mg/L) there was a rapid reduction in S. typhi initially, but the numbers of the organism eventually leveled off and maintained a concentration of 102 cells per ml. The authors reported a decrease in the decimal reduction times between the control {T 90, 4.22 days; VF A concentration, 100 125 mg/L) and experimental (T90, 18.63 days; VFA concentration, 4800 5700 mg/L) digesters. The study, however, did not determine the concentration of specific VF As. Kwon and Ricke (1999) specifically studied the effect of propionate on S. typhimurium as a pure culture under anaerobic conditions. Details of the study are given previously (see Chapter 2). Complete growth inhibition of S. typhimurium was observed at a propionate concentration of 1830 mg/L. Sensitivity to VF As under anaerobic conditions has also been demonstrated with E.coli. Abdul and Lloyd (1985) showed that, under anaerobic

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31 conditions, E. coli was inhibited by increasing concentrations ( 60 to 180 mM) of acetate, propionate, and isobutyrate. The experimental conditions are described previously (see Chapter2). Volatile fatty acid concentrations may contribute to indicator and pathogenic organism decimation during anaerobic digestion. However, during anaerobic digestion, VF As, particularly acetate, serves as substrates for methanogenesis. Sooknah and Wilkie (2004) found that the concentrations of acetate in the effluent of a fixed-film digester, operated at a 2-day HRT and ambient temperature treating flushed dairy manure wastewater was below 170 mg/L or undetectable. Therefore, VF As may not contribute to indicator and pathogenic bacteria reduction during anaerobic digestion in a fixed-film system. The contribution of the indigenous microflora in the liquid phase during fixed-film anaerobic digestion to indicator and pathogenic bacteria reduction may include the production of antimicrobial compounds, more so than the production ofVFAs. However, competition between the indigenous microflora and indicator and pathogenic bacteria may also occur. Thus, the role of indigenous microflora in the liquid phase during fixed film anaerobic digestion remains unclear. Purpose The purpose of the current experiment was to determine if the reduction of indictor and pathogenic bacteria in the liquid phase of fixed film anaerobic digestion at ambient temperature is attributed to the presence of indigenous microflora, inhibition of growth by digester contents, or by starvation.

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32 Materials and Methods Dairy Research Unit The University of Florida Dairy Research Unit (DRU) is located in Hague, FL. The facility has an average milking herd of 500 cows, which are housed and fed in free stall barns. These barns are constructed with slanted concrete floors to allow downward flow of the flush water to remove manure and urine. Sand is used as bedding in the free stall barns instead of organic bedding materials because sand is inorganic and limits bacterial growth. Manure Handling The manure handling process starts with flushing of the free stall barns and the milking parlor. Manure is removed by intermittent, automated flushing. Sand is removed by sedimentation in the sand trap and fibrous solids are removed using a mechanical solids separator. The remaining solids are further removed by sedimentation basins. The resulting slurry (influent) flows through a bar-screen before entering the pump sump. Fixed-fllm Anaerobic Digester The fixed-film anaerobic digester used in the study is a demonstration scale unit with a 100,000 gal total capacity (97,377 gal active liquid volume). The system was operated at ambient temperature and an average HRT of 3-days. The system is located past the free stall barns and adjacent to the wast e storage pond. The digester is equipped with a flare and is used to convert excess methane to CO2 and H20, to avoid methane emissions. Influent is fed into the digester from the pump sump. The material is recycled through inert media to be degraded by the attached biofilm. Anaerobically di ges t ed effluent is stored in waste storage ponds before being applied to land.

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33 Sample Collection, Characterization, and Preparation Sample Collection. Influent and effluent was collected from the DRU. Influent was collected as three 1 L samples from the pump sump. Effluent was collected as three 1 L samples from a sampling port located on the side of the fixed-film reactor. The port is used for talcing samples of the effluent prior to exiting the system. Sample temperature was measured at the time of collection. Characterization. The samples were characterized by measuring the following parameters according to standard methods (APHA, 1998): pH (section 4500-H B), total solids (TS, section 2540 B), volatile solids (VS, section 2540 E), suspended solids (SS, section 2540 D) and volatile suspended solids (VSS, section 2540 E). Total chemical oxygen demand (TCOD) was performed on the whole fraction of samples using COD tubes (Hach Co. Loveland CO). Soluble chemical oxygen demand (SCOD) was performed on the soluble fraction of wastewater. The soluble fraction was obtained by centrifugation at 11, 000 x g for 30 minutes and filtration through a 0.45 m nitrocellulose filter (Millipore) A 5-day biochemical oxygen demand (BOD5 ) was measured on the whole (TBOD5 ) and soluble (SBOD s ) fractions of the samples using a BODTrak apparatus (Hach Co. Loveland, CO) To prevent nitrification, 175 mg 2 chloro 6 trichloromethyl pyridine (N-Serve # 253335, Hach Co., Loveland CO) was added to each BOD bottle Microbial Analysis. The microbial population (i.e. anaerobes aerobes and facultative org anisms) was determined Total anaerobes and aerobes wer e measured by direct platin g onto plate count agar (Difeo) Platin g a nd incubation for total an ae rob e s was perform e d in the anaerobic chamber (described later) All plates were incubated at

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34 room temperature for 5 days. Total facultative organisms were determined by most probable number (MPN) technique using fluid thioglycollate (Difeo) tightly capped and incubated as previously stated using aerobic conditions. Organic acids and sugars were measured in the soluble fractions of influent and effiuent. The samples (100 ml) were ionized by adjusting to pH 11 with 5 N NaOH. The samples were then concentrated using a microrotary film evaporator (EvapotecTM, #4214000, Labconoco, Lenexa, KS) at 100C. Concentrated samples were reconstituted with deionized water, using 3 ml for influent and 2 ml for effiuent. Reconstituted samples were then filtered through a 0.45m nitrocellulose filter (Millipore). The filtered samples were analyzed using a Hewlett-Packard HPLC (HP 1090 Series II) equipped with a refractive index (RI) detector and a UV monitor (210 nm). Bacterial Cultures Escherichia coli, Enterococcus spp., S. aureus, and Salmonella spp. used in the study were isolated previously from influent. Escherichia coli O157:H? (ATCC 43888) was obtained from the American Type Culture Collection. All cultures were grown in 3% (w/v) tryptic soy broth (TSB) overnight at 37C. The cells were harvested by centrifugation at 2,000 x g for 10 minutes and washed three times with sterile IX phosphate buffered saline pH 7.5 (PBS). The cells were resuspended in 10 ml PBS and stored at 5C until use. Bacterial Quantification Bacterial concentrations were determined by direct plate counts using mFC agar mE agar mannitol salt agar (acriflavine added for whole fractions) and X LD agar for E coli (and fe cal coliforms) Ent e rococcus spp., S. aureus and Salmon e ll a s pp. respectively All media was prepared according to the manufacturer s i n structions.

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35 Enterococcus spp. and S. aureus were incubated at 37C for 48 hours. Fecal coliforms and E. coli were incubated at 45C overnight. Salmonella spp. was incubated at 37C for 24 hours. Total anaerobes and aerobes were quantified as described previously. Anaerobic Conditions Anaerobic conditions during incubation were maintained in an anaerobic chamber (Coy, Grass Lake, MD, which was filled with a gas mixture of 80% N2 and 20% CO2 Residual oxygen was removed from the chamber using hydrogen and palladium catalysts. Pipettes and equipment were stored within the chamber to minimize oxygen contamination. The chamber was also equipped with a forced air incubator. Survival in Whole and Soluble Wastewater Fractions The soluble fraction of influent was used to expose the test organisms to the substrates present in the liquid phase during feeding of the digester. The soluble fraction of effluent was used to expose the test organisms to the substrates that would be present in the liquid phase during digestion. The soluble fractions of influent and effluent were obtained as previously described in "Sample Collection, Characterization, and Preparation." The soluble fractions were transferred as 5 ml aliquots in triplicate to sterile, capped 13mm test tubes. The samples were inoculated individually with each test organism, prepared as previously described. The samples were then transferred to the anaerobic chamber and incubated for 3 days at 28C Positive and negative controls were 3% TSB and PBS, respectively. Total anaerobes, aerobes, facultative organisms, and test organisms were measured as previously described. The whole fraction of influent was used to expose the test organisms to the indigenous population that would be present in the liquid phase during feeding of the digester. The whole fraction of effluent was used to expose the test organisms to the

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36 indigenous population that would be present during digestion of the liquid phase. The whole fractions of influent and effiuent were transferred as 5 ml aliquots in triplicate into sterile, capped 13mm test tubes and screw-cap tubes containing a magnetic stirring bar. The tubes were transferred to the anaerobic chamber and incubated for 3 days at 28C (without mixing) and 38C (mixed). Total anaerobes, aerobes, facultative organisms, fecal coliforms, Enterococcus spp., and S. aureus were measured as previously described. The survival of E.coli 0157:H7 and indigenous Salmonella spp. was not studied in experiments with the whole fraction. Due to the low occurrence of these pathogens in the wastestream of the DRU, artificial inoculation would have been required and potentially would hinder simulating the natural population encountered by indigenous fecal coliforms, Enterococcus spp. and S. aureus Inhibition and Wastewater Supplementation Studies Inhibition effects were determined by supplementing the soluble fractions of influent and effiuent with 0.09% TSB. The amount ofTSB used was the minimal concentration that could support the growth of S. aureus. Soluble influent and effiuent, and supplemented soluble fractions were inoculated with a suspension of S. aureus prepared as previously described. Negative and positive controls were PBS and 0.09% TSB, respectively. The samples were transferred to the anaerobic chamber and incubated under for 3 days at 28C. Staphylococcus aureus were quantified as previously described. Effiuent was supplemented with a carbon source and yeast extract (YE) to determine the effect of substrate limitation on the proliferation of S. aureus. Effiuent was supplemented with 5 and 55 mM glucose, 0.1 % YE, and ashed YE. Glucose served as a sole source of carbon and YE served to represent organic carbon and nutrients that may

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37 be present in manure from cellular debris. The use of 55 mM glucose was based on the carbohydrate concentration of commercial growth media (e.g., mannital salt agar, Difeo). The percentages of carbon in 5 mM and 55 mM glucose was equivalent to the percentage of carbon in 0.1 % YE (Table 3-1 ). Ashed YE served as a source of inorganic nutrients and minerals. Ashed YE was obtained by ignition of 5g YE at 550C for 4 hrs. The residue following ignition was added to the effluent at the same amount present in 0.1 % YE. Soluble wastewater, supplemented soluble wastewater, and controls (i.e., PBS, 3% TSB, and influent) were inoculated with S. aureus suspended in PBS The samples were transferred to the anaerobic chamber, incubated, and assayed for S. aureus as previously described. Statistical Analysis Log10 increases or decreases were determined under each condition by subtracting the final log10 transformed concentration from the initial log10 transformed concentration. Each condition was compared with the positive control and negative control using oneway ANOVA and Duncan's Method with a significance level of0.05. The statistical analysis was performed using ProStat v 3.5. Results Wastewater Characteristics The properties of the wastewat e r samples used in the study are given in Table 3-2. Anaerobic treatment reduced TCOD by 52%, SCOD by 72%, TBODs by 62%, and SBOD5 by 57%. Total aerobes, anaerobes, and facultative organisms were reduced by 62%, 73% and 19%, respectively. Acetate and acetoin were reduced by greater than 80%.

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38 Growth of Bacteria in Whole and Soluble Fractions of Wastewater To determine if the indigenous microflora affected the concentrations of indicator bacteria (fecal coliforms and Enterococcus spp.) and pathogenic bacteria (S. aureus, Salmonella spp. and E. coli O157:H7), influent and effluent with and without indigenous microflora (i.e., whole and soluble fractions, respectively) were incubated under anaerobic conditions for 3 days at 28C. A. Total aerobic, anaerobic, and facultative bacteria Soluble wastewater fraction. Growth of total aerobic, anaerobic, and facultative bacteria in the soluble fractions of influent and effluent is shown in Table 3-3. Log 1o increases in concentration of all groups were significantly lower (P < 0 05) in influent than in 3% TSB. However there was no significant difference (P < 0.05) between the log10 increases in concentration between 3% TSB and effluent for all three groups. Furthermore, there was more growth of all groups in soluble effluent than in soluble influent. Although growth of all groups was lower in the influent, the log10 increase for both groups was significantly higher (P < 0.05) than the other organisms used in the study. This trend is also observed with effluent. Whole wastewater fraction. Log10 decreases in concentrations of indigenous total anaerobic, aerobic, and facultative bacteria were observed in the whole fractions of influent and effluent at 38C and 28C {Table 3-4). Unlike tests with the soluble fractions where 10 to 20 l of wastewater was used as an inoculum, the whole fractions were incubated without any alterations. Indigenous concentrations of all groups may have already been at a maximum in the whole fractions, where further incubation resulted in a decline in population. The log10 reduction of total anaerobes betwe e n 38C and 28C in influent was not significantly different (P < 0.05). This trend was aJso observed in the

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39 effluent. The log10 reduction for total anaerobic bacteria was significantly lower (P < 0.05) in effluent at 38C and 28C than in influent at the same teml?eratures. Conversely, the log10 decrease for total aerobic bacteria was significantly lower (P < 0.05) in influent at 38C and 28C and in effluent at 38C than in effluent at 28C. Also, the reduction of total aerobes in influent at 38C and 28C was significantly lower (P < 0.05) than for total anaerobes in influent at the same temperature. The reduction of total facultative organisms at 38C in influent and effluent and 28C in influent was significantly higher than at 28C in effluent. Furthermore, the reduction of total anaerobes and facultative organisms in effluent at 28C was significantly lower (P < 0.05) than for total aerobes, fecal coliforms, Enterococcus spp, and S. aureus {Table 3-5). These results suggest that the anaerobic and facultative population is more stable and active in the effluent. B. E. coli and fecal coliforms Soluble wastewater fraction. Escherichia coli isolated from influent was chosen as a representative of the fecal coliform population in influent and effluent. This organism was able to proliferate in the soluble fractions of influent and effluent {Table 33). However, growth in both fractions was significantly lower (P < 0.05) than in the positive control. Furthermore, growth of this strain of E. coli in effluent was significantly lower (P < 0.05) than in influent. This result shows that effluent is limited in the amount of available substrate to support significant increases in concentration. Whole wastewater fraction. In the whole fractions of influent and effluent, fecal coliforms were not detected after incubation at 38C or 28C (Table 3-4). This result shows that in presence of indigenous total aerobic and anaerobic organisms, fecal coliforms were not able to survive in influent or effluent at either temperature.

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40 C. Enterococcus spp. Soluble wastewater fraction. Enterococcus spp. isolated from influent was able to proliferate in both soluble fractions of influent and effluent (Table 3-3). There was no significant difference (P < 0.05) between the log10 increase in influent and the positive control. However, the log10 increase in effluent was significantly lower (P < 0.05) than influent and the positive control. These results show that anaerobic treatment reduces the amount of available substrate to support significant increases in concentration of indigenous Enterococcus spp. Whole wastewater fraction. There was significantly more reduction of Enterococcus spp. in influent at 38C than at 28C (Table 3-4). Also, there was significantly more reduction in influent at both temperatures than in effluent. The reduction of Enterococcus spp. in effluent at 38C and 28C was not significantly different (P < 0.05) (Table 3-4). There were significantly more reduction of Enterococcus spp in influent at both temperatures than for total aerobes and anaerobes (Table 3-5). In the effluent at both temperatures, the reduction of Enterococcus spp. was significantly higher (P < 0.05) than for total anaerobes (Table 3 5) In the whole fractions of influent and effluent the log10 reductions of Ente ro c o c cus spp. were 1.04 and 0 69 respectively (Table 3-4). This result shows that the indigenous microflora in influent and effluent contributes to the reduction of this organism during the digest ion process. These results also suggest that mesophilic temperatur e s are not required to reduce Enterococcus spp

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41 D. Salmonella spp. Soluble wastewater fraction. Salmonella spp. isolated from influent was able to proliferate in the soluble fractions of influent and effluent (Table 3-3). However, growth in both fractions was significantly lower (P < 0.05) than the positive control. E. E. coli 0157:H7 Soluble wastewater fraction. Escherichia coli 0157:H7 was able to proliferate in the soluble fractions of influent and effluent (Table 3-3). Also, there was no significant difference (P < 0 05) between the log10 increases in concentrations for the positive control, influent and effluent. Differences in growth were observed between pathogenic and nonpathogenic E.coli in the soluble fractions of influent and effluent. Growth in influent was similar to that of indigenous E coli. However the log10 increase in effluent for E.coli isolated from influent was significantly lower (P < 0.05) than for E.coli 0157:H7. F.S.aureus Soluble wastewater fraction. There was no significant difference (P < 0.05) in the growth of indigenous S. aureus in the soluble fraction of influent and the positive control (Table 3 3). There was also no significant difference (P < 0.05) between the lo g10 decreases in effluent and the negative control. This shows that S. aureus can proliferate in influent but not effluent. Whole wastewater fraction. Reductions of S. aureus in influent and efflu e nt at 38 C and 28C were similar (Table 3-4). Also, the reduction of S. aureus in effluent at 38 C and 28C was not significantly different (P < 0 05) These results suggest that dig e stion at both temp e r a tur es r esult in similar d ecre as e s of S au reus in e ffluent.

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42 However, there was significantly more reduction of S. aureus in influent at 38C than at 28C. The reduction of S. aureus in effluent at 38C and 28C was significantly higher (P < 0.05) than for total anaerobes and aerobes (Table 3-~DHowever, the reductions of S. aureus observed in the whole fraction of effluent at both temperatures (-0.84) were less than those observed in the soluble fraction (-1.43) at 28C. These results suggest that either inhibitory compounds or nutritional limitations adversely impact S. aureus. Inhibition or starvation of S. aureus. To determine if inhibitory compounds or substrate limitations impacted S. aureus, soluble effluent was supplemented with a minimal concentration of TSB. As previously observed, S. aureus was not able to proliferate in effluent (Table 3-6). However, supplementation with TSB resulted in a 1.6 log10 increase in concentration. This result suggests that proliferaton of S. aureus is restricted by substrate limitations, not inhibitory compounds. Wastewater supplementation. Supplementation resulted in proliferation of S. aureus in soluble effluent (Table 3-7). Log1o increases of S. aureus in effluent supplemented with 55 mM glucose solution were significantly higher (P < 0.05) than in the other solutions tested. Growth was not observed in effluent or 5mM and 55 mM glucose solutions. However, addition of 5 mM and 55 mM glucose to effluent resulted in proliferation of S. aureus. Also, there was no growth detected in effluent supplemented with ashed yeast extract, suggesting that inorganic nutrition is not the limiting substrate These results demonstrate that the inability of S. aureus to proliferate in the effluent is due to the lack of sufficient sources of carbon.

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43 Discussion and Conclusions Anaerobic digestion is known to reduce the concentrations of pathogenic bacteria from various manures. The decimation is commonly attributed to operating temperature and HRT, where higher temperatures and longer HR.Ts result in higher rates of decimation. However, manure contains a diverse microbial population and the influence of the indigenous micro flora in manure on the survival of indicator organisms and pathogenic bacteria has not been studied extensively. Few studies have investigated the influence of indigenous micro flora on pathogenic bacteria in manure, under anaerobic conditions. Shin et al. (2002) found that a fecal suspension (FS) and its anaerobic culture (FC) displayed inhibitory effects on E coli O157:H7. An overnight culture of E.coli O157:H7 (1 x 105 CFU/ml) was grown in mixed culture with FS (5 x 108 CFU/ml) and FC ( 5 x 108 CFU/ml) anaerobically for 24 h at 3 7C. Growth of these pathogenic bacteria was inhibited in the presence of FS and FC. Also decimation of E. coli and Salmonella spp. during mesophilic anaerobic digestion of sewage sludge (20 day HRT} has been attributed to competition with indigenous microflora (Smith et al. 2005). Similar impacts of indigeous micro flora have be e n demonstrated against S. aur eu s Donnelly et al. (1968) studied the proliferation and enterotoxin production of S. aureus (104 CFU/ml) under aerobic conditions in raw milk with a high (> 106 CFU/ml) and low ( < 104 CFU/ml) standard plate count (SPC, i.e. indi g enous microflora). At 30C S. aureus was able to proliferate and produce toxin in low SPC milk. However, in high SPC milk at the same temperature, S aureus demonstrated less proliferation and no enterotoxin was detect e d The authors concluded that S. aureus was adversely impact e d by the activity o f indig e nous micro flora. In th e curr e nt s tudy th e indigenous microflora in wastewater (107 C FU/ ml) caused decimation of nati ve S. aureus (103 CFU/ml).

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44 DiGiacinto and Frazier (1966) demonstrated inhibited growth of S. aureus during incubation with coliforms and Proteus spp under aerobic conditions in TSB. Eschericihia coli (2 x 104 CFU/ml) and Proteus vulgaris (2 x 104 CFU/ml), both commonly found in animal manure, were found to inhibit the growth of S. aureus (2 x 104 CFU/ml) in mixed cultures at 22C and 30C. Results from the current study showed significant reduction of fecal coliforms (> 2 log10) in the presence of indigenous microflora, suggesting their competition with S. aureus may be minimal. However, competition between S. aureus and Proteus spp. during fixed-film anaerobic digestion is not known. The current study suggests that the indigenous microflora responsible for decimation of indicator and pathogenic bacteria may consist of strict anaerobic and facultative organisms. Total anaerobic and facultative organisms were reduced significantly less than fecal coliforms, Enterococcus spp., and S. aureus. These results suggest that the activity of the anaerobic and facultative population in the liquid phase contributes to the reduction of indicator and pathogenic bacteria during fixed-film anaerobic digestion. In the current study, the test organisms, with the exception of S. aureus, were able to proliferate in the soluble fraction of effluent, whereas growth did not occur in the whole fraction of effluent. The difference in growth may be attributed to the ability of the test organisms, as a pure culture, to use the remaining carbon sources ( e.g., acetate and acetoin). The concentration of the remaining acetate (1.1 mM) and acetoin (0.4 mM) may have been too low to support the proliferation S. aureus

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45 Exposure to optimum growth temperature of bacterial pathogens, in the presence of indigenous microflora, does not increase the proliferation of pathogenic bacteria (Donnelly et al., 1968). Similarity, in the current study, the reductions of indicator and pathogenic bacteria observed in the whole fraction of effiuent at 38C and 28C were similar. These results suggest that anaerobic digestion at optimum growth temperatures of indicator and pathogenic bacteria will not increase survival in the presence of indigenous microflora Limited substrate concentration was found to specifically affect S. aureus. In the current study, S. aureus was the only test organism that could not proliferate in the soluble fraction of effiuent, where growth was observed in the soluble fraction of influent. Furthermore, a higher reduction was observed in the soluble fraction of effiuent than in the whole fraction of effiuent, suggesting that removal of nutrients or the presence of inhibitory compounds, resulting from the activity of indigenous micro flora, influenced reduction of S. aureus during incubation. Supplementation with tryptic soy broth demonstrated that starvation, and not inhibitory compounds, were preventing proliferation of S. aureus. This finding suggests that substrates necessary to support S. aureus were removed during anaerobic treatment of influent, resulting in starvation of S. aureus. Furthermore because S. aureus was the only test organism that did not grow in the soluble fraction of effiuent, the use of the tested indicators to predict the survivability of S. aureus following digestion may not be valid Therefore, direct monitoring of S aureus, as apposed to using surrogates, may be necessary. Starvation of S. aureus during anaerobic di gesti on may be induced by the inability to compete with the indigenous micro:flora The inability to compete with the indigenous

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46 microflora may be attributed to the differences in concentrations in native wastewater. The indigenous population was present at 106 to 107 CFU/ml and S. aureus was present at 102 to 103 CFU/ml. Furthermore, during digestion, there was a low reduction (19%) of facultative organisms, suggesting their stability in the liquid phase during digestion. Thus, S. aureus may have been out competed for available substrates, resulting in starvation. Although S. aureus may be reduced by starvation, survival of the residual population during digestion may be possible. In stressed conditions, S. aureus enters a starvation-survival state. The starvation state of S. aureus was characterized by Clements and Forster (1998). Differential protein expression and morphological changes of S. aureus were observed after the cells were exposed to long-term glucose limiting conditions. The morphological changes associated with the starvation state included reduction in cell size and lack of division septa. Several different proteins not normally observed in vegetative cells were expressed prior to and during the starvation state. This finding suggested that S. aureus utilized differential protein expression to enter the starvation state and that the cells were not dormant during the starvation state Also, during starvation conditions, a 3 log1o reduction of vegetative S. aureus was observed before entry into the starvation state. The investigators found that the recovery of S. aureus from a starvation state could not occur by the addition of solely glucose or amino acids. A combination of glucose and amino acids were required to induce growth. Also, an unknown factor ( or factors) present in media utilized by vegetative S. aureus cells was found to induce growth of starved cells.

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47 Another phenotypic trait of starvation-state S. aureus is the formation of smaller colonies, as compared to normal vegetative cells, that are similar to small colony variants (Clements et al., 1999). Small colony variants of S. aureus are mutations that arise from stressed conditions. The mutations result in decreased coagulase and hemolysin activity, slow growth, reduced or no pigmentation, and small colony size ( diameter < 0.1 mm) on agar media (Clements and Foster, 1999, Kaplan and Dye, 1976). In the current study, the results demonstrated proliferation of S. aureus in the soluble fraction of effluent following the addition of only carbon. This result shows that S. aureus was starved for carbon and that carbon is a growth-limiting constituent in effluent. During anaerobic digestion, carbon is converted mainly to C~, CO2 and biomass. The reduced COD of the effluent demonstrated the reduction of carbon during anaerobic digestion. The reduced COD suggests that utilization of carbon by other indigenous microflora during anaerobic digestion resulted in starvation of S. aureus. Entry into a starvation stated during anaerobic digestion may occur when the organism becomes stressed. The current study shows the reduction of indicator and pathogenic bacteria in the liquid fraction is due to microbial competition and starvation from the lack of sufficient carbon sources These findings suggest possible mechanisms for the decimation of these organisms during anaerobic treatment. The current study showed reductions of these organisms in the presence of indigenous micro flora at ambient temperature (28C) and a low HRT (3 days). The results ofthis study demonstrate the importance of indigenous microflora in manure for sanitization of manure wastewater by anaerobic digestion. Furthermore, removal of carbon sourc es necessary to support pathogenic bacteria may

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48 play a role in decimation. Also, the use of indicators to predict the presence of S. aureus should be reevaluated.

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49 Table 3-1. Percentage of carbon from various sources in yeast extract. o/c t % Carbon from Component* # Carbons Mol. Wt. %Carbon o componen m t YE componen m YE Ala 3 89 40.48 5.36 2.17 Arg 6 174 41.41 3.02 1.25 Cys 3 121 29.78 0.74 0 22 Asp 4 133 36.12 6.69 2.42 Glu 5 147 40.85 14.20 5.80 Gly 2 75 32. 03 3 25 1.04 His 6 155 46.49 1.20 0 .56 Ile 6 131 55 .01 3 .23 1.78 Leu 6 131 55.01 4.69 2.58 Lys 6 146 49.36 5.15 2.54 Met 5 149 40.30 1.05 0.42 Phe 9 165 65.51 2.53 1.66 Pro 5 115 52 .22 2.60 1.36 Ser 3 105 34.31 2.84 0.97 Thr 4 119 40.37 2.95 1.19 Trp 11 204 64.76 1.36 0.88 Tyr 9 181 59 72 1.20 0.72 Val 5 117 51.32 3.79 1.95 carbohi'.drates 6 180 40. 03 17.50 7.01 Biotin 10 244.31 46. 19 0 0003 0.00015 Choline chloride 5 139.63 43.01 0 03 0.013 folic acid 19 441.4 51.70 0.0002 0.00008 Inositol 6 180.16 40.00 0.14 0 056 nicotinic acid 6 123.11 58.53 0.060 0.035 PABA 7 137.00 47. 98 0.076 0.037 Pantothenic acid 9 219 23 49.30 0.027 0.014 Pyridoxine 8 205.64 46.72 0.004 0.002 Riboflavine 17 376.36 54.25 0 012 0.006 Thiamine 12 300.81 42.73 0.053 0.023 th}'Illldine 10 242.23 49.58 0 002 0.0009 TOTAL% C in YE 36.69 Amount (%C) in 1 % YE 0.367 Amount (%C) in 55 mM carbon source 0.390 Amount (%C) in 0.1 % YE 0.0 37 Amount (%C) in 5 mM carbon source 0 036 *The components of yeast extract (YE) were obtained from a typical analysis of YE (Difeo Laboratories, 1998)

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50 Table 3-2. Characterization of samples used for all experiments. Parameter Influent Effluent Temperature, C 24 25 pH 7.55 7.06 TCOD,mg/L 3707 15** 1779 31 SCOD,mg/L 2083 590 TS,mg/L 3904 169 3064 SS,mg/L 1136 69 1034 VS,mg/L 2352 114 1543 40 VSS, mg/L 920 780 65 TBODs,mg/L 3413 175 1313 64 SBODs, mg/L 1046 5 448 Total aerobes, CFU/ml 2.1 0.2 X 107 7.9 1.0 X 106 Total anaerobes, CFU/ml 1.3 0.4 X 107 3.4 1.1 X 106 Total facultative, MPN/ml 9.4 0.3 X 106 7.6 0.5 X 106 Acetate, mM 6.3 0.7 1.1 .07 Acetoin, mM 2.1 0 6 0.42 0 02 Notes: Anaerobically digested flushed dairy manure wasetewater (effluent) was obtained from the University of Florida IF AS fixed-film anaerobic digester Flushed dairy manure wastewater (influent) was material collected prior to anaerobic treatment. *Parameter abbreviations are as follows: TCOD = total chemical oxygen demand, SCOD = soluble chemical oxygen demand, TS = total solids SS = suspended solids, VS = volatile solids, VSS = volatile suspended solids, TBOD5 = 5-day total biochemical oxygen demand and SBOD5 = 5-day soluble biochemical oxygen demand. **Values are average standard deviation (n = 3)

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51 Table 3-3 Growth of indicat o r and path o genic bacteria in the soluble fraction of wastewater. Initial cone, Log10 Reduction Organism CFU/ml 3%TSB lXPBS Influent Effluent Total aerobes 3.6 0.6 X 104 4.l0 A NA 2.6013 3 .9~ Total anaerobes 1.7 0.2 X 104 3.54A NA 2.868 3.63A Total facultative, 8.3 0.3 X 103 3.98A NA 2 .95 c 4.15A MPN / ml E.coli 3.9 0.6 X 106 1.48A -0.54D 0 .748 0.34c Enterococcus 6.3 0.2 X 1 06 0 .54A <-5.00 0.41 A 0 .268 spp. S. aureus 7.9 0 2 X 105 0 .33A 1.438 0 .50A -l.438 Salmonella spp. 2.5 .1 X 106 2.00A <-5.00 0.91 c 1.11 B E c oli O157:H7 6.4 0.3 X 106 0.80A -0.718 0.66A 0.67A *Notes: The soluble fraction of influent and effiuent were collected as described in the text. A small volume (20 l) of influent was used as the inoculum for total aerobic anaerobic, and facultative organisms The filtrate was inoculated with each test organism and incubated without mixing under anaerobic conditions for 3 days at 28C. Concentration values are average standard deviation (n = 3) NA not applicable A D Values with the same letter within the same row are not significantly different (P < 0 05)

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52 Table 3-4. Growth of indigen o us indicat o r b acteria and S aureus in the whole fraction of wastewater. Organism Total anaerobes Total aerobes Total facultative Fecal coliforms Enterococcus spp. S. aureus Influent Initial cone, CFU/ml 1.3 0 4 X 107 2.1 0.2 X 107 9.4 0 3 X 106 2.4 0.3 X 105 7.2 1.2 X 105 1.3 0 6 X 103 38C -0. 5 B C -0.2A -1.0B <-4.0 -l.3 c -0.98 L og10 Reduction Effluent 28C -0 .6c -0.lA -l.4c < 3. 0 -1.0B -0.7A Initial cone, CFU/ml 3.4 1.1 X 106 7.9 1.0 X 106 7.6 0.5 X 106 2.8 0.2 X 104 2.2 1.2 X 105 1.0 0.1 X 102 38C 0.2A -0.lA -1.0B <-3.0 -0.8A -0 .8A,B A-CValues with the same letter in the same row are not significant (P < 0.05) 28C -0.4A, B -0.6B -0.4A <-2. 0 -0 .7A -0.8A,B Notes : Influent and effluent samples were incubated with under anaerobic conditions for 3 days at 38C (mixing) or 28C (static). Total anaerobes aerobes fecal coliforrns Enterococcus spp. and S. aureus were measured as described in the text. Concentration values are average standard deviation (n= 3).

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53 Table 3-5. Comparison of growth between indigenous bacteria in wastewater at 38C and 28C. Log10 Reduction Organism Influent Effluent 38C 28C 38C 28C Total anaerobes -0.SB -0.6B -0.2A -0.4A Total aerobes -0.2A -0.1 A -0.IA -0.6B Total facultative -1.0 c -1.4D -1.0B -0.4A Fecal colifonns <-4.0 <-3.0 <-3.0 <-2.0 Enterococcus spp. -1.3D -1.0c -0.8B -0.7B, C S aureus -0.9c -0.7B -0.8B -0.8c A 0values with the same letter in the same column are not significant (P < 0 05) Notes: Influent and effluent samples were incubated under anaerobic conditions for 3 days at 38C (mixing) or 28C (static) Total anaerobes, aerobes, fecal coliforms, Enterococcus spp., and S. aureus were measured as described in the text.

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54 Table 3-6. Determination of inhibitory or nutritional limitation on the growth of indigenous S. aureus. Initial cone, Final c o ne, Log10 Log10 c o ntribution of Condition mcrease or CFU/ml CFU/ml (decrease) 0.09%TSB 0%TSB 3.4 0.3 X 104 1.3 0.1 X 103 -l.4F 0.09%TSB 6.1 0.5 X 103 1.0 0.2 X 107 3.2A Influent 2.1 0.4 X 104 4.4 0.4 X 105 1.3D Influent+ 1.7 0.2 X 104 4.3 0.7 X 106 2.48 1.18 0.09%TSB Effiuent 1.5 0.3 X 104 2.5 0.2 X 103 -0.8E Effiuent + 1.6 0.4 X 104 6.4 1.6 X 105 1.6c 2.4A 0 .09%TSB Notes: The soluble fraction of influent and effluent were collected by centrifugation at 11,000 x g for 30 mins. The supernatant was filtered using a 0.45 mm nitrocellulose filter. The filtrates were supplemented with tryptic soy broth (TSB) to a final concentration of0. 09%. The filtrates, supplemented filtrates, and lX PBS (0% TSB) were inoculated with S. aureus isolated from influent and incubated without mixing under anaerobic conditions for 3 days at 28C. Samples were assayed daily for test organisms Concentration values are average standard deviation (n= 3). A Fvalues with the same letter within the same column are not significantly different (P < 0 05).

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55 Table 37 Growth of S. aureus in soluble effiuent and soluble effluent with various amendments. Condition Initial cone, CFU/ml Final cone, CFU/ml Log increase or decrease lXPBS 1.1 0 .15 X 104 5.2 1.2 X 103 -0.5G 3%TSB 9 7 1.5 X 103 5.4 l.0x 107 3.38 Influent 1.2 0.25 X 104 3.1 0.38 X 107 2.4 Effluent 7.0 2.0 X 103 2.8 0 3 X 103 -0.4G Glucose 5 mM 9.7 1.2 X 1 03 4 8 3 2 X 102 -l.4H Effluent + glucose 5 mM 9.7 1.2 X 103 7.7 1.5 X 104 0 9 E Glucose 55 mM 6.0 1.3 X 103 2 2 0 53 X 103 -0.4G Effluent+ gluc o se, 55 mM 1.6 0.31 X 104 9.5 0.33 X 107 3 .6A Yeast Extract, 0.1% 2 6 0.36 X 104 2.3 0 93 X 107 2 5 c Effluent + Yeast Extract, 0 1 % 2 9 0.15 X 104 2 4 0.66 X 107 2 9 c Ashed Yeast Extract 3.2 0.7 X 104 1.6 0 2 X 104 -0.3 G *Yeast extract at 0 1 % is equivalent to 5 mM glucose on a carbon basis (see Table 3-1 ). Notes: Phosphate buffered saline (lX PBS) 3% tryptic soy broth (TSB) influent effluent, supplemented effluent, and supplement solutions in sterile saline were inoculated with S aureus suspended in PBS. Samples were incubated without mixing under anaerobic c onditions for 3 days at 28C. Concentration values are average standard deviati o n (n= 3). A H Values with the same letters within the same column are not significantly different (P < 0.05).

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CHAPTER4 INDICATOR AND PATHOGENIC BACTERIA REDUCTION DURING ANAEROBIC DIGESTION: ATTACHMENT TO THE FIXED FILM Introduction Fixed-bed anaerobic digesters are systems that use internal support media where anaerobic bacteria attach to the surface and proliferate, resulting in the formation of a biofilm (see Chapter 2). The retained biofilm (i.e. fixed-film) allows for a higher treatment capacity as compared to systems that do not use internal support media (Ramasamy and Abbasi, 2000). The increased treatment capacity for systems with internal support media is due to the retention of methanogens, which grow at a slow rate (Wilkie and Colleran, 1989). Digesters that are not constructed with an internal support media can potentially lose a substantial portion of the methanogenic population as effiuent exits the system. To maintain treatment efficiency, such systems have to operate at long retention times (> IO days) to reestablish a methanogenic population. Pathogen reduction by anaerobic digestion has been well studied in various systems, including a fixed-film system (Barnes, 2002, Davis et al., 2001, Hill, 2003). However, for fixed-film systems, the role of the retained biofilm in pathogen reduction during anaerobic digestion remains unclear. Purpose The purpose of the current study was to use a model organism to determine if indicator and pathogenic bacteria were removed from the liquid phase during anaerobic digestion by attachment to the fixed-film. 56

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57 Materials and Methods Pilot-Scale Fixed-Film Reactors The two pilot-scale fixed-film reactors used for biofilm attachment studies had a total capacity of I 04 gal each. Both reactors were operated at ambient temperature (28C to 33C) and a 3-day HRT. The systems were considered at steady state once SCOD reduction was consistent (69% 5%). Both reactors were packed with vertically orientated PVC pipes to provide internal support media for biofilm formation. Each system was continuously fed influent from a feeding manifold by a peristaltic pump (MasterFlex). However, one unit was operated in up-flow mode, where contents entered the reactor from the bottom and the other was operated in down-flow mode, where contents entered the reactor from the top. Contents of both reactors were mixed, by recycling at a 3: I ratio to feed rate, by another peristaltic pump. Bacterial Culture Escherichia coli BL(2 l )DE3 haboring plasmid pl.AMI 055, for the production of green fluorescent protein (GFP), was grown at 37C overnight in LB broth, Lennox (Fisher) with 0.4 mM isopropyl /3-D-1-thiogalactopyranoside (IPTG) (Amresco, Solon, OH) and 15.4 g/ml of ampicillin (Sigma). The cells were harvested by centrifugation at 2,000 x g for 10 minutes and washed three times with sterile IX PBS. The cells were resuspended in 100 ml IX PBS and stored at 5C until use. Survival Comparability with Indigenous Fecal Coliforms Influent and eflluent samples were collected as previously described (see Chapter 3). GFP producing E. coli were inoculated into whole fractions of influent and eflluent. Samples were incubated under anaerobic conditions (see Chapter 3) for 3 days at 28C. Indigenous fecal coliforms were assayed daily as previously described (see Chapter 3).

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58 GFP producing E. coli were assayed daily using plate count agar supplemented with 0.00002% crystal violet (Carolina Biological Supply Co., Burlington, NC), 0.4 mM IPTG, and 15.4 g/ml ampicillin. Following incubation, GFP producing E.coli colonies were enumerated by exposing the plates to a UV light source (366 nm). Biofllm Attachment Studies A 100 ml suspension of GFP producing E. coli (1.2 x 107 CFU/ml) was prepared as previously described. The suspension of bacteria was fed into the reactor through the feeding pumps. Effluent samples from the reactor were collected at 1 h intervals over 12 h, and then a composite sample was taken daily. Serial dilutions of eflluent samples were done using IX PBS. Viable counts of GFP producing E. coli were performed by direct plating diluted samples onto plate count agar prepared and incubated as previously described. Total counts of GFP producing E. coli were performed by filtering diluted samples through a 0.45 m polycarbonate filter (47 mm, Nucleopore Corp., Pleasanton, CA). Filters were mounted on glass slides (50 mm x 75 mm) and cells were enumerated under 250X total magnification with a fluorescent microscope (Carl Zeiss, Standard 25) equipped with a UV light source (470 nm). The total number oftest organisms expected to be present in the effluent at each sampling event was calculated by the following: Predicted effluent volume= (reactor volume/ HRT) x sampling time interval Predicted effluent CFU = (initial CFU I reactor volume) x predicted effluent volume Biof"llm Sampling and Examining Support media in close proximity to where influent entered each system were chosen for sampling Biofilm samples were obtained by removing selected support media from each reactor. Then, 2 in sections were cut from the bottom (up flow reactor) and top (down flow reactor) of the support media Each 2 in section was then divided into

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. ~~---------------59 smaller sections and placed inside containers lined with moist paper towels to prevent desiccation. The sections were examined under 300X total magnification with a stereomicroscope (Leica MZ7s) equipped with a UV light source (470 nm). Photos of the samples were taken using a digital camera (Nikon Coolpix 4500) connected to the microscope by a lens attachment (Martin Microscope mmcool, SIN: 0941). Results Fecal Coliform and GFP E. coli Survival The GFP producing E. coli displayed comparable survival with indigenous fecal coliforms in both the influent and effluent, suggesting that the test organism could serve as a suitable model for indigenous fecal coliforms (Figure 4-1 ). Biofdm Attachment Studies Each pilot-scale fixed-film reactor was fed a suspension of GFP producing E coli. The number of cells detected at each sampling event in the up-flow reactor was lower than the predicted values (Figure 4-2). There was a 1 log10 difference between the predicted values and the experimental values at each sampling event. Also, the model organism could not be detected on Day 4 and after Day 6. After 1 HRT (3 days), there was a 0.9 logio reduction of the model organism. During the first 12 h, retention of the model organism in the down-flow reactor was similar to the upflow-reactor (Figure 4-3). However on Day 1, 4 and 6 the experimental values were similar to the predicted values. The model organism was not detected aft e r Day 7. After 1 HRT there was a 0.2 log10 reduction of the model organism. To confirm the attachment of the model organism to the fixed-film biofilm samples w e r e obtain e d and e xamined GFP producing E coli w e re det e ct e d on th e fixed film (Figure 4 4) However, the viability of the attached organisms was not determined.

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60 Discussion and Conclusions The current study demonstrates the role of the fixed-film for reducing indicator and pathogenic bacteria during digestion. Our results show that the test organism attached to the fixed-film, thereby reducing effluent concentrations of the test organism. However, a difference in retention was observed between the two modes of operation. After 3 days of operation, the reduction in the up-flow mode (0.7 log10) was higher than in the down flow mode (O.l log10). Conversely, Barnes (2002) reported a 0.2 log1oreduction of fecal coliforms, using the same reactor operated in up-flow mode at the same conditions as the current study. Also, in the study, a similar reduction (0.4 log10 ) of fecal coliforms was achieved during operation in down-flow mode. However, in the current study, the difference between the two modes suggests that operation in up-flow mode may lead to more interaction between the fixed-film and indicator and pathogenic organisms. Anaerobic systems that employ fixed-beds have demonstrated higher treatment efficiencies than systems without fixed-beds. However, the contribution of the fixed-film to pathogen reduction from various substrates has not been extensively studied. The current study provided evidence that the fixed-film contributes to the reduction of indicator and pathogenic bacteria during anaerobic digestion. After 3 days of operation, a 0.7 logio reduction was achieved. Previously, microbial competition was shown to account for more than 2 log1o reduction of indicator bacteria in batch studies (see Chapter 3). However, the attachment studies were performed with a continuously fed system, where substrates and organisms were constantly introduced. In continuously fed systems, microbial competition may still have a significant contribution to indicator and pathogen reduction, but the constant influx of substrates and organisms may decrease this effect, in comparison to the batch studies. However, in the current study, model organisms

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61 detected in the effiuent were not viable. This result suggests that inactivation of the model organism occurred in the liquid phase during digestion. Previous results (see Chapter 3) suggest that the inactivation in the liquid phase observed in the current study may be due to microbial competition. In a continuously fed system, the fixed-film provides a physical means for removal, whereas microbial competition may inactivate unattached organisms in the liquid phase. The combination of attachment to the fixed film and microbial competition may synergistically contribute to the reduction of indicator and pathogenic bacteria during digestion in the fixed-film reactor.

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62 1.2 0 .8 A 0 0.6 u 0.4 0.2 0 1.2 0 8 ~FC ---GFP B 0 u ""' 0 6 u 0 4 0 2 0 0 2 3 Time, day Figure 4 -1. Survival of indigenous fecal coliforms and GFP producing E. coli in wastewater. Notes: Influent (A) and effluent (B) were inoculated with GFP producing. coli suspended in PBS. Samples were incubated under anaerobic conditions for 3 days at 28C. Fecal coliforms ( D ) and GFP producing coli (D ) were assayed daily as described in the text. Each point is an average and error bars represent standard deviation (n = 3).

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63 l.0E+09 l.0E+08 l.0E+07 l.0E+06 II ... s I.0E+05 u l.0E+04 I.0E+03 I.0E+02 l.0E+0l l.0E+OO _,._ Predicted concentration -0Experimental concentration -&Predicted I logl O reduction 0.08 0.13 0.17 0.21 0.38 0.42 0.46 0.5 Time, days 3 4 6 7 8 9 Figure 4-2. Retention of GFP E.coli within the pilot-scale fixed-film anaerobic digester operated in up-flow mode. Notes: A pilot-scale fixed-film reactor was fed a suspension ofGFP producing E coli. Effluent samples were collected and total counts of GFP producing E. coli were performed as described in the text. Each point represents triplicate samples and error bars represent standard deviation

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64 l.0E+09 l .0E +08 l.0E+07 1.0E+06 r: l.0E+05 u 1.0E+04 l.0E+03 -+Predicted concentration l.0E+02 -0Experimental concentration l.0E+0l _,._ Predicted 1 logl O reduction l.0E+00 0.08 0.13 0.17 0.21 0.38 0.42 0.46 0.5 1 3 4 6 7 8 9 Time, days Figure 4-3. Retention of GFP producing E. coli within the pilot-scale fixed-film operated in down-flow mode. Notes: A pilot-scale fixed-film reactor was fed a suspension of GFP producing E coli Effluent samples were collected and total counts of GFP producing E. coli were performed as described in the text. Each point represents triplicate samples and error bars represent standard deviation

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65 Figure 4-4. GFP producing E. coli attached to the surface of the biofilm. Notes: Samples of internal support media were removed from the pilot-scale fixed-film reactor. Sampl ~s were cut from support media and viewed under 300 X total magnification with a UV light source (470 nm) White arrows indicate GFP producin g E coli.

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CHAPTERS BACTERIOPHAGE REDUCTION DURING ANAEROBIC DIGESTION: THE ROLE OF INDIGENOUS MICROFLORA Introduction Animal manure may contain pathogenic viruses that pose severe human and herd health concerns (Bicudo and Goyal, 2003, Pell, 1996). Therefore, treatment of manure is necessary to reduce the levels of these organisms Anaerobic digestion can effectively reduce the concentration of viruses from various substrates, including manure. Berg and Berman (1980) found that mesophilic and thermophilic anaerobic digestion (20 day HRT) of domestic sludges achieved a 1 log 1o and 3 log 1o reduction, respectively, of viruses after 20 days. Similarly, Huyard et al. (2000) investigated virus reduction in sludge by a two-phase anaerobic digester {TP AD) operated at thermophilic (2 day HRT) and mesophilic (10 day HRT) temperature. The authors reported a 4 log1o reduction of poliovirus after 10 days. Also, Aitken et al. (2005) demonstrated that thermophilic anaerobic digestion, at a 4 day HRT and treating sludge, achieved > 4 log10 reduction of poliovirus. However, reduction of male-specific bacteriophages was 1 to 2.6 log10. Spillman et al. (1987) reported inactivation rates ofless than 1 log10 daf1 for viruses during mesophilic anaerobic digestion of sludge. Inactivation rates during thermophilic digestion varied between 0.2 to> 8 log10 hr -1 However the authors did not report the HRT of the digester used in the study. Virus reductions similar to the aforementioned studies have been found during treatment of animal manure Lund et al. (1996) found that mesophilic anaerobic 66

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67 digestion (15 day HRT) of mixed animal manure (25% pig, 75% cow) achieved a 4 log10 reduction of bovine enterovirus and porcine parvovirus after 1 and 2 days, respectively. Derbyshire et al. (1986) examined the occurrence of viruses in pig manure following mesophilic anaerobic digestion. The authors detected viruses during the first 5 months of the study. However, the authors did not report virus concentrations, virus reduction rates, nor the HRT of the digester. Treatment of dairy manure wastewater by fixed-film anaerobic digestion at a 3 day HRT and ambient temperature (28C), can achieve a 1 log1o reduction ofbacteriophages and viruses (Barnes, 2002, Davis, 2001). Previous studies on viral inactivation by anaerobic digestion point reductions to operational parameters (i.e. temperature and retention time) However antagonism by indigenous manure microflora has been suggested to play a critical role in inactivating viruses during anaerobic digestion (Deng and Cliver 1995, Ward, 1982). Therefore the role of indigenous microflora in reducing virus during anaerobic digestion should be investigated. Purpose The purpose of the current study was to determine if the reduction of viruses in flushed dairy manure wastewater during fixed film anaerobic digestion at ambient temperature is attributed to the presence of indigenous manure micro flora. Materials and Methods Sample Collection, Characterization, and Preparation Flushed dairy manure wastewater (influent) and anaerobically treated flushed dairy manure wastewat e r (effluent) were collected from the University of Florida Dairy R e search Unit (DRU) in Hague, F L and charact erize d a s pr e viously described (see Chapter 3)

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68 Bacteriophages Bacteriophages and their respective hosts were MS2 (ATCC 15597-Bl) and E.coli C-3000 (ATCC 15597), X174 (ATCC 13706-B5) andE. coli (ATCC 13706), and PRDl and Salmonella Typhimurium (ATCC 19585). Experimental Design Whole and soluble fractions of influent and effluent were prepared as previously described (see Chapter 3). Soluble fractions were inoculated with each organism. The samples were transferred to the anaerobic chamber and incubated for 3 days at 28C. Groundwater collected from the DRU was used as a negative control for experiments using bacteriophages Bacteriophage Quantification Bacteriophages were enumerated as plaque forming units per ml (PFU/ml) using the soft agar overlay technique (Hurst, 1997). Anaerobic Conditions Anaerobic conditions during incubation were maintained in an anaerobic chamber as previously described (see Chapter 3) Statistical Analysis Log10 increases or decreases were determined under each condition by subtracting the final log10 transformed concentration from the initial log1o transformed concentration Each condition was compared with the positive control and negative control using one way ANOVA and Duncan's Method with a significance level of 0.05. The statistical analysis was performed using ProStat v 3.5.

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69 Results Wastewater Characteristics Characteristics of the wastewaters used for experiments are given in Chapter 3 (see Table 3-2). Influence of Whole and Soluble Fractions of Wastewater To determine if the indigenous microflora affected the concentrations of bacteriophages, influent and effluent with and without indigenous microflora (i.e., whole and soluble fractions, respectively) were inoculated with approximately 105 PFU/ml of bacteriophages. The samples were incubated under anaerobic conditions for 3 days at 28C. In the soluble fractions of influent and effluent, bacteriophages (MS2, PRDl and Xl 74) were stable throughout the duration of the experiment, and were not significantly different (P > 0.05) from those observed in groundwater (Table 5-1). However when incubated in the presence of indigenous micro flora, there were significant reductions (P < 0.05) in the concentrations of each bacteriophage in both influent and effluent (Table 5-1 ) There was a significantly higher reduction (P < 0.05) ofMS2 and Xl 74 in effluent than in influent. The highest reductions were observed with PRD-1 However the reductions of PRD 1 observed in influent and effluent were not significantly different (P < 0.05). These results show that the activity of indigenous microflora in influent and effluent can contribute to bacteriophage inactivation under anaerobic conditions and ambient temperature. Discussion and Conclusions The current study demonstrates the role o f indigenous microflora on inactivation o f viruses during anaerobic digestion. All viruses st udied were stable in soluble fractions o f wastewater incubated under anaerobic conditions at 28C for 3 days. However in the

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70 presence of indigenous microflora, all the viruses studied were reduced by greater than 1 log10. Previous studies have shown inactivation of viruses during mesophilic and submesophilic (15 to 25C) temperatures may be attributed by microbial antagonism. Pesaro et al (1995) conducted field studies to investigate virus inactivation in liquid cattle manure stored under anaerobic conditions using membrane sandwiches with and without pores. At submesophilic temperatures, decimation times for viruses were significantly lower for membranes with pores than those without. The results suggest that microbial products may contribute to inactivation of viruses. Ward (1982) investigated antiviral activity of microorganisms in activated sludge (mixed-liquor suspended solids MLSS) against poliovirus 1 under aerobic conditions. The study demonstrated that viruses were inactivated in the presence ofMLSS and in broth previously incubated with MLSS. These results suggested that microbial products could contribute to inactivation of viruses during anaerobic treatment. Deng and Cliver (1992) found similar inactivation of poliovirus 1 by bacteria in unmixed, stored swine manure. The authors reported that decimation times in manure incubated under aerobic conditions at 37C and 25C were significantly less than those in cell-free controls at the same temperatures. A later study by the same authors suggest that microbial mediated inactivation of viruses may not always be enzymatic (Deng and Cliver, 1995). Production of antiviral substances that are not enzymatic (virolytic substances) has been demonstrated with Pseudomonas aeruginosa (Cliver and Herrmann, 1972). The current study provides further evidence of the critical role indigenous microflora have during fixed-film anaerobic digestion. Although microbial processes

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71 during anaerobic digestion are shown to reduce virus concentrations, complete removal is not achieved Reductions of greater than 3 log10 may be achieved, but as demonstrated by previous studies either longer retention times or higher operating temperatures are necessary to allow for more reduction. However, operations desiring to minimize energy input to digesters must balance performance with wastewater production, thereby sacrificing extensive virus removal. Therefore, effluents from digesters will inherently contain residual concentrations of active viruses, which may be introduced to the environment during land application.

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72 Table 5-1 Impact of indigenous micro flora on viruses suspended in groundwater and wastewater. Log10 Reduction Organism Groundwater Influent Efiluent Soluble Whole Soluble Whole Soluble Whole fraction fraction fraction fraction fraction fraction MS2 -0.58 -0.53 -0.2A -0.8b 0.0 -1.3b PRDl -0.3A -0.3a -0.lA -2.5b -0.2A -2.4b X174 -0.5A -0.53 -0.2A -l.2b -0.3A -I.Sc Notes : Groundwater influent, and effluent were prepared as described in the text (s e e Chapter 3). Samples were inoculated with viruses and incubated under anaerobic conditions at 28C for 3 days. Each value is an average log1 0 reduction (n = 3). A B a-cvalues with the same letter within the same row are not significantly different (P < 0 05)

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CHAPTER6 TRANSPORT OF VIRUSES IN SOIL AMENDED WITH ANAEROBICALLY DIGESTED FLUHSHED DAIRY MANURE WASTEWATER Introduction Intensification and concentration of large dairy operations have led to issues with manure management. Two major concerns are nuisance odors and possible contamination of groundwater resources following land application of manure. In an attempt to reduce these problems, a fixed-film anaerobic digester has been constructed at the University of Florida's Dairy Research Unit (DRU). Freestall barns at the DRU are hydraulically flushed with water to remove animal manure. The fixed-film digester then treats the flushed dairy manure wastewater (influent) before it is land applied for forage crop production. Anaerobic digestion significantly reduces the COD of flushed dairy manure (Wilkie, 2005). Previous studies have shown that this unit can also reduce the levels of indicator and pathogenic bacteria and bacteriophages by approximately 90% (Davis et al 2001). However, the use of fixed-film anaerobic digesters on flushed manure is a relatively new technology and the fate of viruses following land application of anaerobically digested flushed dairy manure ( effluent) is unknown Virus Inactivation during Anaerobic Digestion Viruses are known to be associated with and survive in animal manure (Elliott and Ellis 1977 Mawdsley et al., 1995, Pesaro et al. 1995). However, anaerobic treatment has been shown to inactivate viruses. Anaerobic digestion at ambient temperature (25 to 31 C) and a 3-day HRT has been shown to achieve a 1.5 log10 reduction of somatic and 7 3

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74 male-specific bacteriophages from flushed dairy manure wastewater (Davis et al., 2001). Lund et al.(1996) found that mesophilic (35C) anaerobic digestion of swine and cow liquid manure required 9 days to achieve a 4 log10 reduction (99.99%). Under thermophilic (55C) conditions, the system required 6 days to achieve a 4 log10 reduction of porcine parvovirus. Anaerobic digestion is effective for reducing the load of viruses in animal manure before land application. However, residual viral concentrations can be detected in eflluents of anaerobic systems treating animal manure and be detected in soil where wastewater has been applied (Derbyshire, 1976, Derbyshire et al., 1986) Although anaerobic digestion achieves significant reductions of viruses from animal manure, residual concentrations of viruses can potentially lead to groundwater contamination following land application of eflluents. Viral Attachment to Soil Viruses in water utilize two mechanisms for attaching to soil, hydrophobic and electrostatic interactions. Physical and chemical parameters that affect virus adsorption to soil include the pH of the aqueous media and the soil, flowrate through the soil matrix, soil organic matter, percent clay, and ionic strength of the suspending medium (Goyal and Gerba, 1979, Kinoshita et al, 1993 Lance et al., 1976, Zhuang and Jin, 2003). pH and isoelectric point. Previous studies have investigated transport, adsorption and survival of viruses in wastewater following land application of wastewater. Several environmental factors have been found to influence viral adsorption in soil. Organic matter and exchangeable iron are inversely correlated with viral adsorption to soil (Gerba and Goyal 1981 Hurst et al., 1980). However, one of the most important factors is pH (Goyal and G e rba 1979). In general, an increase in soil pH will result in a decrease in viral adsorption whereas a decrease in soil pH will increase viral

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75 adsorption. However, environmental factors, including pH, do not influence the adsorption of all viruses to the same degree. Gerba and Goyal (1981) examined the differences in adsorption among viruses and categorized the viruses into two groups based on factors that contributed to adsorption. The factors that were most important for adsorption of group I viruses, which included Xl 74 and MS2, to soil were pH, organic matter content, and exchangeable iron. However, group II viruses, which included poliovirus 1 LSc and coxsackie B3, soil particle surface area was the only significant variable that contributed to adsorption. The impact of pH on viral adsorption is related to the isoelectric point (pl) of the virus (Dowd et al., 1998). The isoelectric point is the pH at which the virus has no net electrical charge. In general, viral adsorption will occur in soils with a pH lower than that of the pl of virus. Viruses and soil particles generally have a net negative charge at a neutral pH At low pH values, viruses acquire a net positive charge resulting in adsorption to soil. At high pH values, viruses acquire a net negative charge and adsorption to soil is minimal and results in elution or desorption of the virus from the soil. The adsorption effects observed with viruses are the result of alte r ations to the proteins on the surface of the virion during pH changes; whereas changes to the charge on soil particles are minimal (Berg 1987 Dowd et al. 1998) Hydrophobic interactions. The side chains of several amino acids found on the prote i n coat of viruses are nonpolar and attract other nonpolar compounds or groups. Hydrophobic bonding occurs when nonpolar groups aggregate Viruses with coats that ar e pr e dominat ely composed of hydrophobic proteins will tend to u se hydrophobic interactions Such viruses include MS 2 and echovirus 5 (Shields and F arrah 2002)

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76 Futhennore, viruses that contain host-derived phospholipid membranes, such as PRDl, will also utilize hydrophobic bonding (Hanninen et al., 1997). Hydrophobic interactions may be demonstrated when the pH of an aqueous medium is adjusted to the pl of the virus. At that point, hydrophobic interactions may predominate (Berg, 1987). Electrostatic interactions. Electrostatic interactions occur between charged side chains of amino acids on viral protein coats and charges on surfaces. Shields and Farrah (2002) compared viral adsorption by electrostatic and hydrophobic interactions. Using DEAE-Sepharose, a solid with charged groups, virus adsorption was characterized by elution from the solids with solutions increasing in ionic strength. Several viruses, including poliovirus 1 LSc and T4, were shown to utilize strong electrostatic interactions. In aqueous media, such as natural waters, the pH is usually neutral and above the pl of most viruses. In such instances, the charge of the viron and soil matrix will be the same and electrostatic interactions will depend on the concentration of cations present with in the aqueous media or the soil. Cations may interact with both the viron and soil surface generating a salt bridge, resulting in adsorption (Berg, 1987). Purpose Viruses have been shown to be inactivated during anaerobic digestion (Davis et al., 2001, Lund et al., 1996). However, residual concentrations of viruses may be introduced to the environment during land application of treated wastewater. To date, the adsorption of viruses to soil following land application of effluent from a fixed film anaerobic digester treating flushed dairy manure wastewater has not been studied. The purpose of the current study is to investigate the adsorption and survival of viruses in flushed dairy manure wastewater and in anaerobically digested wastewater to soil.

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77 Materials and Methods Collection and Analysis of Soil Samples Soil was collected from the DRU in Hague, FL, in an area mapped as typic Quartzipsamments; sandy soil with greater than 90% having particle size between 0.02 to 2.0 mm (USDA, 1999). Samples were taken at 0.8 m depth from a sprayfield that receives effluent from a tertiary short-term storage pond. Soil samples were thoroughly mixed to yield a composite sample. The soil was air dried at room temperature and sieved. Batch adsorption studies were performed on soil with particle size less than 0.2 mm. To increase the flow rate through columns, soil with particle size of0.2 to 0.8 mm was used for column studies. Soil pH and conductivity were measured as described by Mylavarapu and Kennelley (2002). Soil particle size analysis was conducted on the whole, batch, and column soil samples. Collection and Analysis of Wastewater, Groundwater, and Rainwater Samples One liter samples of influent and effluent were collected from the DRU manure management facility Grab samples of influent were collected from a wet well prior to anaerobic treatment. Effluent was collected from a port located on the fixed-film anaerobic digester After settling, supernatant fractions of both wastewaters were used for experiments. Groundwater was collected from wells located at the DRU and rainwater was collected during storm events. Sample pH and conductivity were measured according to standard methods (Standard Methods, 1998). Viruses and Viral Assays Bacteriophages and their hosts used in this study were MS2 (ATCC 15597-B1), host E.coli C-3000 (ATCC 1 5597); Xl 74 (ATCC 13706-B5), host E coli (ATCC 13706); and PRDl, host Salmon e lla Typhimurium (ATCC 19585) Phages were

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78 quantified as plaque-forming units (PFU) using the agar overlay technique, as described by Hurst (1997). Poliovirus 1 LSc (ATCC VR-59) was assayed as PFU on Buffalo green monkey kidney cells, as described by Hurst (1997). Characteristics of the viruses used in the study are given in Table 6-1. Virus Stability The stability of viruses in groundwater and wastewater was tested. During all batch and column studies, viruses were suspended in groundwater and wastewater and assayed at the beginning and end of each experiment to check for inactivation during an experiment. Viral assays were performed as previously described. Attachment and Detachment Studies Batch studies Batch adsorption studies were performed with groundwater, influent wastewater, and effluent from the anaerobic digester. The study was designed using nine batches so that each experimental condition was performed in triplicate. The entire experiment was performed twice to yield a total of six observations for each experimental condition. Two ml samples of groundwater, influent, or effluent were inoculated with viruses and added to 1 g soil samples (particle size< 0.2 mm). Samples were mixed on a reciprocating shaker at 55 rpm for 1 h, followed by centrifugation at 3,000 x g for 10 min. The supernatant fraction was collected and assayed. The soil pellets were mixed with 10 ml 3% beef extract (BE), pH 7, for 30 min to recover adsorbed viruses (Hurst, 1997). The samples were centrifuged again and the supernatant fraction was collected and assayed for viruses as previously described To determine the influence of groundwater, influent, and effluent on desorption of enteroviruses and bacteriophag es from soil, 1 g soil samples were set up as previously described. Each soil sample was mixed with 2 ml of inoculated groundwater for 1 h and

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79 centrifuged as previously described. The supernatant fraction was assayed and the soil pellets were treated with 10 ml of either groundwater, 3% BE, influent, or effluent for 30 min. The samples were centrifuged at 3,000 x g for 10 minutes and the supernatant fractions were assayed Column studies. Adsorption studies with soil columns were performed using 45 g of soil (0.2 to 0.8 mm) packed in 60 ml syringes with a borosilicate fiberglass filter in the bottom of the syringe. Each experimental condition was performed in triplicate. One pore volume (20 ml) of groundwater, influent, or effluent was inoculated with viruses and passed through the columns at 1 ml/min. The percolate was collected and assayed to determine the percentage of viral adsorption. Each column was then removed from the syringes and placed in a 500 ml centrifuge bottle. From each column, 5 g was removed, dried and then mixed with 10 ml of deionized water for 2 h to measure soil pH. The remaining 40 g of soil was mixed for 30 min with 40 ml 10% buffered BE (100 g/L BE, 13.4 g/L sodium phosphate dibasic 1.2 g/L citric acid) (Hurst, 1997). The samples were centrifuged at 5,000 x g for 10 min and then the supernatant fraction was collected and assayed. To study potential mobilization of viruses by influent and effluent, one pore volume of groundwater inoculated with viruses was added to each column. The percolate was collected and assayed to determine the percentage of viral adsorption. Six pore volumes of rainwater was added to each column. The percolates were collected and assayed for viruses. An additional six pore volumes of either rainwater, influent, or effluent was passed through the columns. The percolates were collected and assayed for

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80 viruses. The columns were removed from the syringes and mixed with BE and assayed for recovered viruses as described previously. Attachment and Detachment Mechanisms Soil COD retention. The amount of soluble organics retained by the soil, expressed as soil soluble chemical oxygen demand (soil SCOD), was calculated by subtracting wastewater initial SCOD from SCOD of the wastewater after percolating through the soil column. The percentage of viruses mobilized at each pore volume was plotted against soil-retained SCOD for the corresponding pore volume and analyzed by linear regression. Influence of pH on virus adsorption. A 100 ml sample of either groundwater, influent, or effluent was adjusted to pH 3.5 with HCI. Phages were added to the adjusted groundwater and wastewater samples. Inoculated samples (2 ml) were added to 1 g of soil samples(< 0.2 mm) and mixed on a reciprocating shaker at 55 rpm for 1 h. The samples were centrifuged, assayed, and the viruses were recovered as described previously for batch studies. Wastewater fractionation. Influent and effluent samples were fractionated using cellulose ester dialysis tubing (Spectra/Poi Biotech). Groundwater, influent, and effluent samples (20 ml) were transferred to dialysis tubing with rolecular weight cut offs (MWCOs) of 100 kDa, 10 kDa, and 1 kDa. Groundwater samples were dialyzed against 1 L influent for 3 days at 4C. Influent and effluent samples ~ere dialyzed against 2 L deionized water for 3 days at 4C. A larger volume of deionzed water was used for dialyzing wastewater samples to remove as much of the target sized compounds as possible. The samples were removed and a 10 ml aliquot of each sample was

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81 inoculated with viruses. Adsorption to soil, virus assays, and virus recovery was performed as previously mentioned for batch studies. Influence of detergents on adsorption and desorption. Adsorption studies were performed using a cationic detergent (hexadecyltrimethylammonium bromide, HT AB, Sigma), anionic detergent (sodium dodecyl sulfate, SOS, FisherBiotech) and a nonionic detergent (polyoxyethylene sorbitan monooleate, Tween 80, Fisher Scientific) were added to groundwater, influent, and effluent samples to a final concentration of0.01 %. The samples were inoculated with viruses and adsorption, assay, and recovery was performed as previously described for batch stu~ies. Batch soil samples (1 g, particle size< 0.2 mm) were pretreated with groundwater supplemented with HTAB, SOS, or Tween 80 to a final concentration of0.01% to determine the impact on virus adsorption to soil. Batch samples were mixed with 10 ml of groundwater detergent solution for 1 h on a reciprocating shaker table as previously mentioned. The samples were centrifuged as described earlier for batch studies and the supernatant was discarded. The pellet was washed three times with 10 ml groundwater to remove any detergent not retained by the soil. To the final soil pellets, 2 ml of groundwater inoculated with viruses was added. The samples were mixed, centrifuged, assayed, and the viruses were recovered as previously described for batch studies. The influence of detergents on desorption of viruses attached to soil samples was performed by adsorbing viruses in groundwater to soil as previously described for batch soil adsorption studies Groundwater was supplemented with HT AB, SOS, or Tween 80 to a final concentration 0.01 %. The soil samples with attached viruses were mixed with 10 ml of detergent solution for 30 min, centrifuged, and assayed as previously described

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82 for batch soil studies. A secondary elution was performed with 3% BE as previously described for batch soil studies. Virus Survival in Soil The survival of viruses in groundwater and wastewater attached to soil was determined. Groundwater, influent, and effluent were inoculated with MS2, PRDl, Xl 74, and poliovirus 1 LSc at 1 x 107 PFU/ml. Inoculated solutions were added to soil as previously described for batch soil studies. Samples were set up in triplicate for each time point. Once a week, viruses were recovered with 3% BE and assayed as previously described for batch soil studies. Risk Assessment of Flushed Dairy Manure A risk assessment was performed to determine the probability of infection following exposure to groundwater where influent and effluent was applied to land. The following exponential dose-response model was used to estimate the risk of infection: 1t = 1 10 -(d/k) where, 7t is the probability of infection, d is the amount of exposure to the organism, and k is the infective dose (Rose and Hass, 1999). Infectious dose data for adenovirus (PHAC, 1999), rotavirus (Graham et al, 1987), and Norwalk virus (Schaub and Oshiro, 2000) was used for the risk assessment. Statistical Analysis The percentage of viruses eluted was calculated by dividing the amount of viruses in the eluent by the amount of viruses adsorbed to soil. The mean percentages of viruses eluted and the differences in viral adsorption betwe e n natural and detergent-pretreated conditions were anal yze d by Student's t-test with a significance level of 0 05. A

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83 comparison of the percentage of viruses adsorbed in groundwater and various virus characteristics (i.e. isoelectric point, type of interaction, and structure) was subjected to regression analysis to determine correlations. Regression analysis was al~o used to determine the effect of soil SCOD retention on virus mobilization in soil columns. All analysis was performed using ProStat v3 5 Results Soil Characteristics The soil used in the batch and column experiments differed in composition (Table 6-2). Soil used for batch and column studies differed in the amount and type of sand. The amount of silt and clay, the reactive species in soil, was higher in the soil used for batch studies than in the column studies. Batch Studies The adsorption of viruses in groundwater influent and effluent to soil are shown in Figure 6-1. When the viruses were suspended in groundwater, there was greater adsorption of 4>Xl 74 (> 99 %) and poliovirus 1 (96 %) than ofMS2 (72 %) and PRDl (48 %) (Table 6 3). In influent, adsorption decreased to 17 % for MS2 and < 1 % for PRDl. The presence of influent also decreased the adsorption of 4>Xl 74 (78 %) but had little effect on the adsorption of poliovirus 1 (93 %). The adsorption ofMS2 (45 %) and PRDl (8 %) was less in effluent than in groundwater but greater than in influent. The adsorption of 4> X l 7 4 and poliovirus 1 was similar in influ ent and e ffluent. The s e r es ults indicate that the adsorption of viruses was influenced by the presence of effluent and influent and by the type of virus. The phages a d s o r b e d to s oil in the pre s ence o f groundwater w e r e deso r b e d by influent (Table 6 4 ) The percent adsorption to soil observed for MS2 and PRDl in

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84 groundwater was lower than for initial adsorption experiments. This difference may be due to variable soil composition during subsampling. The percentage of all phages detached by influent was significantly higher (P < 0.05) than the percentage detached by effluent or groundwater. The percent eluted by either effluent or groundwater was not significantly different (P < 0.05) for MS2 and PRDl. Poliovirus 1 adsorbed to the soil was not eluted by groundwater, influent or effluent. These results show that, except for poliovirus 1, untreated wastewater can detach more adsorbed phages from soil as compared with anaerobically treated wastewater Column Studies The adsorption of viruses in groundwater, influent and effluent to soil columns is shown in Figure 6-2 The adsorption pattern for the viruses studied was similar to that observed in the batch studies in that the adsorption of cl>Xl 74 (97 %) and poliovirus 1 (99 %) in groundwater was greater than the adsorption ofMS2 (88 %) and PRDl (54 %). In contrast with the batch studies, the difference between adsorption ofMS2 and PRDl in the presence of influent and effluent was small. As observed in the batch studies, influent was found to mobilize adsorbed viruses (Table 6-5). Mobili z ation ofMS2, PRDl and cl>X l 74 by influent was significantly higher (P < 0.05) than by effluent or rainwater Influent mobilized 2 % ofpoliovirus 1 whereas no mobili z at ion was observed with effluent o r rainwater Conductivity and pH The initial pH of the soil samples was considerably lower than the pH of groundwat e r rainw a t e r influent and effluent sampl e s (Table 6-6). In both th e batch and column experim e nts th e r e was an increase in p H in a ll samples following mi x in g o f soil with one of the solut i ons described above. However, we found that normali z ing the pH

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85 of the soil to pH 7.0 during the experiment did not affect adsorption and elution (Table 6-7). Furthermore, during percolation, conductivity, or ionic strength, of the soil samples decreased with rainwater and increased with influent and effluent (Table 6-6). Attachment and Detachment Mechanisms Soluble organics. MS2 elution displayed a positive correlation with soil SCOD (r = 0.936) (Figure 6-3). PRDl (r = 0.103) and Xl 74 (r = 0.037) displayed a positive, but low correlation with soil SCOD. Wastewater fractionation. To determine the size of the compound(s) in influent and effluent affecting virus adsorption to soil dialysis was used to remove compounds smaller than 100 kDa, 10 kDa, and 1 kDa from the wastewaters Viruses were inoculated into the wastewater samples that retained compounds larger than the indicated MWCOs. In samples of influent where molecular weight fractions larger than 10 kDa were retained adsorption ofMS2 (Figure 6-4) was sim i lar to adsorption in groundwater for batch studies (Figure 6-1 ). This result suggests that compounds in the influent that were smaller than 10 kDa were mainly responsible for interfering with adsorption of MS2 to soil. Adsorption did not significantly increase in the influent where compounds larger than 1 kDa were retained. However, adsorption ofMS2 to soil was higher in the effiuent where compounds larger than 1 kDa were retained as compared with adsorption with groundwater in batch studies These results sugg e st that in the influent removal or alteration of compounds between 10 kDa and 1 kDa int e rferes with adsorption ofMS2 to soil. The ad s orption of PRDI to soil was impact e d b y different fractions of influ e nt an d effluent (Figure 6 5) In the effluent, retention o f compounds larger than 10 kDa did not

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86 interfere with adsorption of PRDl to soil. In the influent, retention of compounds larger than 100 kDa did not interfere with adsorption of PRD 1 to soil. These results suggest that compounds in the influent, between 100 kDa and 10 kDa, affect adsorption of PRD 1 to soil. The adsorption pattern of Xl 74 in fractionated wastewater to soil is shown in Figure 6-6. In both the influent and effluent, retention of compounds larger than 100 kDa did not interfere with adsorption of X 17 4 to soil. There were no significant differences observed in adsorption between native wastewater and wastewater retaining compounds larger than 10 kDa and 1 kDa. This finding suggest that compounds in the influent, between 100 kDa and 10 kDa, interefere with adsorption of Xl 74 to soil. Influent compounds smaller than 100 kDa and 10 kDa were transferred to groundwater, by dialyzing 20 ml of groundwater with 100 kDa and 10 kDa MWCOs against 1 L of influent Viruses were inoculated into groundwater, dialyzed groundwater, influent, and influent used for dialysis. The samples were mixed with soil as previously described for batch studies. Adsorption patterns for PRDl and Xl 74 are shown in Figure 6 7 Adsorption of PRDl and Xl 74 to soil decreased when suspended in groundwater containing influent compounds smaller than 100 kDa. Adsorption of PRDl in influent and groundwater dialyzed with influent to soil was similar. Adsorption of Xl 74 to was lower using suspensions of groundwater dialyzed with influent than in raw influent. Furthermore a slight increase in adsorption was observed for both viruses suspended in influent used for dialysis. These results along with the results from Figures 6 5 and 6 6 d e monstrate the interference of influent compounds smaller than 100 kDa on adsorption of PRDl and Xl 74 to soil.

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87 The influence of influent compounds smaller than 10 kDa on adsorption of MS2 to soil was also performed. MS2, suspended in groundwater containing influent compounds smaller than 10 kDa, was not detected in the supematants after mixing with the soil for 1 h. Furthermore, MS2 was not detected in the initial stock solutions of groundwater containing influent compounds smaller than 10 kDa. To determine if influent compounds smaller than 10 kDa inactivated MS2, groundwater and groundwater dialyzed against influent was inoculated with MS2. A 300 l aliquot was taken at 10 min intervals for 1 h. Influent compounds smaller than 10 kDa were found to inactivate MS2 (Figure 6 8). Inactivation was not observed in experiments using the whole fraction of influent. This may be due to interaction between particulate organics and the compounds causing inactivation, thereby preventing inactivation of MS2. Influence of isoelectric point on virus adsorption to soil. Adsorption of viruses in groundwater to soil in batch studies correlated with isoelectric point (r = 0.973) (Figure 6-9). Influence of pH on virus adsorption to soil. To determine the effect wastewater had on virus adsorption at pH values below the pl, the pH of the groundwater and wastewaters were adjusted to 3.5. Adsorption to soil by all three viruses in groundwater and wastewater adjusted to pH 3 5 increased to > 99 % (Table 6-8) Hydrophobic and electrostatic interactions. Viruses utili z e h y drophobic and electrostatic interactions to bind to soil. To determine the type of interactions used by the viruses under study ionic and nonionic detergents were used. Ionic and nonionic detergent s w e r e add e d to groundwater and wast e water to a final concentration of0.01 %, the maximum concentration where viruses rema i ned active. The detergent solutions were

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88 inoculated with viruses and batch adsorption studies were performed as previously described. All three detergents increased the adsorption of MS2 and PRD 1 suspended in groundwater to soil (Figure 6-10). However, adsorption ofXl 74 in groundwater decreased in the presence of Tween 80, was unaffected by SDS, and increased to greater than 99% in the presence ofHTAB. The same pattern of adsorption to soil was seen when detergents and viruses were added to influent (Figure 6-11 ). Again, all three detergents increased the adsorption of MS2 and PRDl to soil. The detergents had little to no effect on adsorption ofXl 74 to soil. The three detergents in effluent had little effect on the adsorption ofMS2 and Xl 74 to soil (Figure 6-12). All three detergents in effluent increased the adsorption of PRD 1 to soil. The effect of ionic and nonionic detergents on virus detachment from soil was determined in batch studies first adsorbing viruses in groundwater to soil. Next, the soil samples, with adsorbed viruses, were mixed with groundwater and groundwater with 0.01 % of each detergent. The detergents eluted less than 40% of the adsorbed viruses in all tests. Detachment of MS2 and Xl 74 from soil by Tween 80 and SDS was not significantly different (P < 0.05) (Table 6-9). Detachment of PRDl by Tween 80 was significantly higher (P < 0.05) than the ionic detergents. Mixing the soil with HTAB in groundwater did not result in detachment of any viruses. Remaining attached viruses were recovered by mixing samples with 10 ml 3% BE for 30 min. The percentage of viruses recovered during the secondary elution are shown

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89 in Table 6-10. Recovery ofMS2 and PRDl with 3% BE from soil batches receiving Tween 80 as the primary eluent was significantly higher (P < 0.05) than batches receiving SDS and HT AB as the primary eluent. There was no significant difference (P < 0.05) between the recoveries of Xl 74 from soil batches receiving Tween 80 and SDS as the primary eluents Recovery of all viruses was lowest from batches receiving HT AB as the primary eluent. To determine if the detergents influenced virus adsorption by interacting with soil binding sites, soil samples were pretreated with groundwater or groundwater with 0.01 % detergent. Viruses in groundwater were mixed with pretreated soil samples in batch studies. Adsorption of MS2 to soil pretreated with Tween 80 and SDS decreased (Figure 6-13). Adsorption of PRDl and Xl 74 to soil pretreated with Tween 80 decreased, but was unaffected in soil pretreated with SDS. Adsorption by all viruses to soil pretreated with HT AB was greater than 90 % The effect of each pretreatment was determined by calculating the difference in adsorption between unaltered soil and pretreated soil. Pretreatment with Tween 80 caused a significant decrease (P < 0.05) in adsorption ofMS2 and PRDl to soil (Table 610). Adsorption ofX174 to soil pretreated with Tween 80 and SDS was not significantly different (P < 0 05) Pretreatment with HT AB increased adsorption of all viruses > 95 % Recovery of viruses from soil pretreated with detergents is shown in Table 6-10. Recovery ofMS2 and PRDl was significantly higher (P < 0.05) from soil pretreated with Tween 80 than from soil pretreat e d w ith SDS and HTAB. Recovery o f MS2 and PRDI

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90 was lowest from soil pretreated with HTAB. Recoveries ofXl 74 from soil were not significantly different (P < 0.05) between detergent pretreatments. Risk Assessment of Viruses Following Land Application Infectious dose data for adenovirus (PHAC, 1999), rotavirus (Graham et al., 1987), and Norwalk virus (Schaub and Oshiro, 2000) was used to determine the probability of infection following exposure to groundwater where influent and eflluent are applied to land. Viruses present in influent applied to land had an 85% probability of causing an infection following exposure to groundwater contaminated with influent (Table 6 -11 ). However, following digestion, the probability of infection dropped to 12%. Furthermore, there was an 88% probability of acquiring an infection from groundwater where influent was land applied resulting in mobilization of viruses attached to soil; whereas application of digested wastewater reduced the probability to 5%. Survival of Viruses in Soil Survival of viruses suspended in groundwater, influent, and effluent applied to soil is shown in Figure 6-14. MS2, PRDl, and Xl 74 decreased during week 1 in all solutions and were not detected thereafter. Poliovirus 1 LSc showed the highest decrease in effiuent during week 1 as compared with groundwater and influent. After week 2, decline of poliovirus 1 LSc was similar for all three solutions. Poliovirus 1 LSc was not detected after week 4. Discussion and Conclusions The adsorption of viruses to soil and virus transport through soil columns has been reported in several studies (Chu e t al. 2 003 McLeod et al., 2001 Quanrud et al., 2003 Z huan g and Jin, 2 003). F actor s that influ e nc e virus adsorption includ e th e char acte ristics

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91 of the viruses, the properties of the soil, and the chemical composition of the solution that contains the viruses. In the batch studies, we have confirmed that the isoelectric point of viruses influences their adsorption to soil (r = 0.973). Also, the presence of untreated wastewater intereferes with virus adsorption to soil. Further more untreated wastewater promotes mobilization of viruses adsorbed to the soil columns. For MS2, mobilization or detachment from soil is influenced by the amount of soluble organics. This result is consistent with characteristics of Group I viruses, where adsorption is influenced by pH and the presence of organics (Gerba and Goyal, 1981). However, the correlation with soluble organics retained by the soil and mobilization of Xl 74, another Group I virus, was low suggesting that other factors in the soil or wastewater interferes with adsorption. The viruses studied demonstrated varying adsorption and elution behavior. Changes in pH of suspending solution soil columns, and natural soils have been shown to influence the adsorption and elution behavior of viruses (Goyal and Gerba, 1979, Kinoshita et al., 1993). In the current study, increases in pH of the inoculated wastewaters were observed during the experiment. However, the observed pH increases did not influence virus adsorption and elution behavior. Additionally, when the pH of the suspending solutions was adjusted below the pl of all the viruses tested, adsorption for all viruses to soil was greater than 99% The presence of dissolved ions in the soil may also influence adsorption and elution of viruses (Hurst et al., 1980). In this study, the conductivity of the soil decreased when mixed with rainwater and increased when mixed with influent and effiuent. Increased mobilization of phages was observed with influent as compared with effiuent. However,

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92 the conductivity of the soil mixed with effiuent was slightly higher than influent. Furthermore, removal of salts from the wastewater during dialysis experiments using 1 k:Da MWCO dialysis tubing did not increase adsorption. Therefore, changes in ionic strength did not appear to be the predominating factor contributing to the mobilization patterns of phages observed in the current study. Compared with untreated wastewater, certain fractions of treated and untreated wastewater resulted in increases in virus adsorption to soil. Retention of compounds larger than 10 k:Da in the influent yielded the most dramatic increase in adsorption of MS2 to soil, as compared to unaltered influent. Transferring influent compounds smaller than 10 k:Da into groundwater caused inactivation of MS2. The results from the current study show that influent compounds smaller than 10 k:Da inactivates MS2. Inactivation was not observed in unaltered influent samples. This may be due to interactions with organics by the virus or the inhibitory compounds, interfering with the inhibitory affect. Retention of compounds larger than 100 k:Da in the influent resulted in increased adsorption of PRDl and Xl 74 to soil, as compared with unaltered influent. Transferring influent compounds smaller than 100 k:Da into groundwater caused decreased adsorption to soil for both viruses, as compared to unaltered groundwater. The adsorption of PRO 1 to soil was similar when viruses were suspended in influent and in groundwater containing influent compounds smaller than 100 k:Da. Adsorption of both viruses to soil increased slightly when suspended in influent where compounds smaller than 100 k:Da were lost to groundwater. The current study shows that influent compounds smaller than 100 k:Da int e rfere with adsorption of PRDl and Xl 74 to soil.

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93 Ionic and nonionic detergents were used to determine the influence of hydrophobic and electrostatic interactions for adsorption to soil by the viruses under study. Groundwater and wastewater with a final detergent concentration of0.01 % impacted the adsorption of added viruses to soil. Tween 80 in groundwater resulted in the greatest decrease in adsorption ofXl 74 to soil as compared with Tween 80 in influent and effluent. However, Tween 80 increased adsorption ofMS2 and PROI. As demonstrated by retention of soluble COD by soil, the soil can react with soluble organics. Interaction between Tween 80 (nonionic detergent) and the soil is also possible, which would result in decreased virus adsorption to soil. However in the current study, adsorption to soil increased for MS2 and PRO 1 in wastewater containing detergents. Alternatively, pretreatment of soil with Tween 80 caused a decrease in adsorption of MS2 and PRO 1. Furthermore, Tween 80 was able elute MS2 and PRDI attached to soil. Adsorption sites may have become unavailable during pretreatment with detergents. Also, hydrophobic and electrostatic interactions between soil and attached viruses may have been interrupted upon introducing Tween 80 to the medium. The effect of soil pretreatment and elution with Tween 80 demonstrates the variable interactions utilized by MS2 and PRDI. Soluble organics in untreated wastewater may bind to available sites on soil. As sites become unavailable for binding, adsorption to soil may be achieved through hydrophobic or electrostatic interactions During anaerobic digestion, the concentration of soluble organics is decreased, which may lead to a decrease in the interference of adsorption by hydrophobic interactions. Furthermore, for PRDI, hydrophobic and some weak electrostatic interactions with the soil may be disrupted by the presence of a specific

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94 soluble component(s) of the influent smaller than 100 kDa, leaving the virus to utilize weak hydrophobic or electrostatic interactions for adsorption to soil. The use of anionic and cationic detergents in groundwater and wastewater, inoculated with viruses, increased virus adsorption to soil. The use of SDS (anionic) in groundwater and wastewater increased the adsorption ofMS2 and PRDl to soil. The viruses and SDS have a net negative charge, which may have resulted in charge repulsion from the detergent in solution. Conversely, pretreating and eluting with SDS caused decreased adsorption and detachment, respectively. The effect ofSDS was more pronounced for PRD 1 in both cases than for MS2. This result further suggests that these viruses may use either hydrophobic or electrostatic interactions to bind to soil. The use ofHTAB (cationic) increased adsorption and was unable to elute any of the viruses used in the study. The presence of cations is known to increase virus adsorption to soil (Goyal and Gerba, 1979, Hurst et al., 1980) Our results show that in the presence ofHTAB, viruses increased adsorption to soil by greater than 95 % and that HT AB did not cause detachment of adsorbed viruses. The survivability of viruses in groundwater, untreated, and treated wastewater was demonstrated in the current study. The phages only remained active for 1 week following inoculation whereas poliovirus 1 LSc persisted for up to 4 weeks. This result suggests that some retained viruses may persist in the soil for prolonged periods. However the current study demonstrated how anaerobic treatment of flushed dairy manure wastewater causes less interference and less desorption of viruses. Therefore, during prolonged periods active viruses may be retained in the soil following application of treated wastewater.

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95 The current study provided evidence demonstrating the adsorption and desorption behavior of selected viruses. In both the batch and column studies, untreated wastewater was shown to decrease viral adsorption to sandy soil. These results suggest that viruses present in untreated dairy manure wastewater may have potential for groundwater contamination during land application. Furthermore, untreated wastewater was shown to increase the release of viruses attached to soil. Thus, land application of untreated dairy manure wastewater may increase the movement of retained viruses through soil, potentially contaminating groundwater. The present study demonstrates the benefit of anaerobic treatment in regards to reducing potential groundwater contamination by viruses in sandy soils. Anaerobic treatment alters the physicochemical characteristics of flushed dairy manure wastewater (Wilkie et al., 2004). The use of untreated wastewater may cause migration of viruses through the soil matrix by interfering with adsorption mechanisms. As demonstrated with MS2, untreated wastewater may disrupt interactions between the soil and MS2 due to the increased amount of soluble organics contributed by untreated wastewater to the soil. Furthermore, influent compounds smaller than 10 kDa was shown to cause inactivation ofMS2. Untreated wastewater also interfered with adsorption of PRDl and Xl 74. This interference is accomplished by component(s) smaller than 100 kDa in the wastewater Anaerobic digestion lowers the soluble organic content and may also remove the fraction interfering with adsorption, resulting in increased virus retention and decreased virus mobilization in the soil. We conclude from the current study that anaerobic treatment of flushed dairy manure wastewater increases the retention of some

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96 viruses to soil and decreases the transport of some adsorbed viruses through the soil matrix, thereby decreasing the potential for groundwater contamination.

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97 Table 6-1 Characteristics of viruses used in the study. Family pl Diureter Nocleic Acid L.,n Interactionb Gro14>c Virus Structure mn content,% MS2 Levivnlae 3 9 24 icosahedral sis(+) strand 0 Strong h}wophobic/ I RNA weak electrostati': PRD-1 Tectiviidae 4.2 63 corrplex JilC8rd/s 16 H)dropbobic/ ND* DNA Electrostati': Cl>Xl74 Muovrilae 6 6 25 27 icosahedral cn:ular sis 0 weak h}wopoobic & (+)DNA electrostati': Poli>viu; l Picomavrilae 4.5 & 30 icosahedral sis ( + ) strand 0 electrostati': D LSc 7.5 RNA *ND Not determined Dowd, S. E. et al. (1998) b Shields, P.A and S R. Farrah. (2002) c Gerba C. P. and S. M Goyal (1981).

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98 Table 6-2 Characteristics of test soil. Test soil for experiment Parameter Whole soil Batch Column Sand, 2 -1 mm(%) 0 1 0 0 0.0 Sand 1 0.5 mm(%) 5.2 0 0 6.3 Sand, 0.5 0.25 mm(%) 41.9 0.1 52 9 Sand, 0.25 0.1 mm(%) 41.7 40.4 35.0 Sand, 0.1 0.05 mm(%) 5 4 40.0 1.2 Sand, total (%) 94.3 80.5 95.4 Silt (%) 2 8 15. 2 3.2 Clay,(%) 2.9 4.3 1.4

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99 Table 6-3 Adsorption of Viruses in groundwater and wastewater to soil. Virus Adsorption to soil, % Groundwater Influent Effluent MS2 72A 17 lOC 458 PRDl 48A 28 88 X174 100 0A 78 148 81 16A, B Poliovirus 1 96A 93 lA 93A Values are average standard deviation (n=6). Notes: Solutions were groundwater ; influent (flushed dairy manure wastewater) and effluent (anaerobically digested flushed dairy manure wastewater) A -evalues with the same letter within the same row are not significantly different (P < 0.05).

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100 Table 6-4 Elution of viruses adsorbed to soil. Virus Initial amount, Adsorbed, Eluted, % of adsorbed PFU % GW Influent Effluent 3%BE MS2 3.2 0.4 X 104 44 41 26 B 100 0 A 50 12 B 89 23 A PRDI 1.1 .1 X 106 33 16 33 128 97A 37 10 B 100 0 A 4lX174 I.I .1 X 104 99 0.2 8 92 A 21 5 C 478 Poliovirus 1 5.1 1.9 X 104 99 0.5 0 0 0 37 1 LSc Notes: Viruses in 2ml of groundwater (GW) were mixed with 1 g of soil for 1 hr. The samples were centrifuged and the supernatant fraction was assayed for viruses. The soil pellets were mixed with 10 ml groundwater, influent, effluent, or 3 % beef extract (BE) for 30 min. The samples were centrifuged and the supernatant fraction assayed to determine the percentage of virus elution. Values are average standard deviation {n = 6). A-CValues with the same letter within the same row are not significantly different (P < 0.05).

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101 Table 6-5 Elution of viruses adsorbed in soil columns. Virus Initial amount, Adsorbed, Eluted, % of adsorbed PFU % Rainwater Influent Effluent 10%BE MS2 7.6 1.6 X 103 91 1 1 0.6 C 63 13 A 16 3 B 61 lOA PRDl 2.4 0.3 X 105 50 21 7 C 79 14 A 36 78 408 ~X174 1.4 0.2 X 105 98 0.3 10 5 B 48 20 A 58 43A Poliovirus 1 1.8 0.1 X 104 99 0.1 0 2. OS 0 41 9A LSc Notes: Viruses were inoculated into 1 pore volume (20 ml) of groundwater and added to soil columns The percolate was collected and assayed to determine the percentage of virus adsorption Six pore volumes of rainwater was added to each column. The percolates were collected and assayed for viruses. Each column then received six pore volumes of eith e r rainwater influent, or effluent. The percolates were collected and assayed to determine the percentage of viruses eluted. An additional column received 1 pore volume of 10% beef extract (BE) to serve as a control. Values are average standard deviation (n= 3) A-CValues with the same letter within the same row are not significantly different (P < 0 05).

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102 Table 6-6 Conductivity and pH changes in soil during batch and column experiments. Sample EH Conductivity, !!Siem Batch Column Column Soil 5.02 5.02 473 Groundwater 7.71 7.71 353 Groundwater+ Soil 6.86 6.50 452 Rainwater NA 7.12 39 Rainwater + Soil NA 5.81 122 Influent 7.77 7.33 3370 Influent + Soil 7 .27 8.04 628 Effluent 7 .20 7.49 3600 Effluent + Soil 7.41 7.82 700 Notes: Influent, flushed dairy manure wastewater ; Effluent anaerobically digested flushed dairy manur e wastewater ; NA, not applicable.

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103 Table 6-7. Adsorption of viruses in groundwater and wastewater to soil adjusted to pH 7.0. Solution Virus Initial amount, Groundwater Influent Effluent PFU Adsorbed, Eluted, % of Adsorbed, Eluted, % of Adsorbed, Eluted, % of % Initial % Initial % Initial MS2 4.1 0.6 X 106 63 36 10 17 10 39 25 PRDl 1.7 0.6 X 105 35 16 8 3 3 9 4 I>X174 1.8 0.5 X 103 91 74 10 78 14 62 10 80 12 69 11 Poliovirus 1 5.7 0.4 X 103 98 0.4 75 89 12 73 11 93 12 74 13 LSc Notes: Batch adsorption studies and elution with 3% beef extract were performed as described in the text. The pH was maintained at 7 .0 during the experiment. Values are average standard deviation (n=3)

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104 Table 6-8 Influence of pH on adsorption of viruses to soil. Sample pH Adsorption, % MS2 PRDl X174 7.68 82.9 6.6 56.6 4.4 99 0.7 Groundwater 3.50 99.8 0 100 99.2 0.9 7.75 27.7 3.2 6.7 4 76.5 1.9 Influent 3.50 99.9 0 100 99.9 0 7.24 42.9.8 16.3 3.4 84.8 1.6 Effluent 3.50 99.7 0.1 100 99.9 Notes: Groundwater influent and effluent were inoculated with viruses. Groundwater and wastewater adjusted to pH 3.5 with 1 N HCl were inoculated with viruses Samples were mixed with soil as described for batch studes. Adsorptioli values are average standard deviation (n = 3).

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105 Table 6-9 Elution of viruses adsorbed to soil by detergents in groundwater. Eluted, % of Adsorbed Adsorbed, Virus 0.01 % Tween 80 0.01% SDS 0.01%HTAB % of Initial Detergent 3%BE Detergent 3%BE Detergent 3%BE MS2 89 12 A 53 151 8A 38 12b 0 10c PRDl 46 30A 401 18 B 25 b 0 8c X174 86 10A 20b 7A 381 0 4 le Notes: Viruses in groundwater were absorbed to 1 g soil samples. A cationic (hexadecyltrimethylammonium bromide, HTAB), anionic (sodium dodecyl sulfate, SDS), and nonionic (polyoxyethylene sorbitan monooleat, Tween 80) detergent was added to groundwater to a final concentration of0.01 %. The detergent solutions were mixed with the soil samples and the percentage of viruses recovered determined. A B Values with the same letter within the same row are not significantly different (P < 0.05). Values are averages standard deviation (n = 3)

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106 Table 6-10 Recovery of viruses with 3% BE from soil pretreated with detergents. Soil Pretreatment Virus Initial amount, 0.01% Tween 80 0.01% sos 0.01%HTAB PFU Adsorbed, Eluted,% of Adsorbed Eluted, % of Adsorbed, Eluted, % of % Adsorbed % Adsorbed % Adsorbed MS2 9 9 1.9 X 104 53 97 A 66 768 95 1 52 12c PRDl 3.8 0 5 X 105 20 91 A 39 11 528 96 1 36c ll>X174 8.3 1.5 X 105 77 34 A 81 30 18A 96 1 349A Notes: Soil samples (1 g) were pretreated with detergents and mixed with viruses as prev iously described. Attached viruses were recovered by mixing soil samples with 3% BE for 30 min. A-CValues with the same letter within the same row are not significantly different (P < 0.05) Values are averages standard deviation (n = 3).

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107 Table 6-11 Estimated risk of infection from exposure to groundwater following land application of wastewater. Influent Etlluent Probability of infection, % Virus introduced by Virus mobilized by wastewater 85 12 wastewater 88 5 Notes: Infectious dose data for adenovirus (PHAC 1999) rotavirus (Graham et al 1987), and Norwalk virus (Schaub and Oshiro, 2000) was used calculating probability of infection. Probability of infection are based on adsorption characteristics of MS2 in batch studies

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108 100 D Groundwater Influent 80 ~Effuent *-(i() -d' Q) ,e 0 20 < I 0 MS2 PRDl PhiXl 74 Poliovirus 1 Figure 6-1 Adsorption of viruses in groundwater and wastewater to soil. Notes: Two ml of groundwater, flushed dairy manure wastewater (influent) and anaerobically digested manure wastewater (effluent) samples were seeded with virus and mixed with 1 g of soil for 1 hr. The samples were centrifuged and the supernatant fraction was assayed to determine the percentage of virus adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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109 D Groumwater .Influent ~Effluent 100 80 ';f_ -d' 60 ] 40 fl) .5 > 20 0 MS2 PRDl PhiX174 Poliovirus 1 Figure 6-2 Adsorption of viruses in groundwater and wastewater to soil columns. Notes: Viruses inoculated into one pore volume (20 ml) of groundwater, flushed dairy manure wastewater (influent), and anaerobically digested flushed dairy manure wastewater (effluent) was added to soil columns The percolates were collected and assayed to determine the percentage of virus adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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110 100 .. r=0.936 80 -= ~(i() :s = I 40 20 0 500 1000 1500 2000 2500 3000 Soil SCOD, mg/L Figure 6-3 Correlation between soluble chemical oxygen demand retained by the soil and MS2 detachment from soil columns. Notes: Soluble chemical oxygen demand (SCOD) retained by the soil after exposure to wastewater was calculated by subtracting initial wastewater SCOD from SCOD of the wastewater after percolating through the soil column. Soil SCOD was determined for each pore volume where viruses were detected in the percolate. Error bars represent the standard deviation (n = 3) of the percentage ofMS2 mobilized through the soil column.

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111 100 D Influent 80 c:I 0 .:: Eftluent e, 0 60 "' 'C < ':le. 0 40 20 0 Natural > 100 kDa > lOkDa > 1 kDa Fraction retained in wastewater Figure 6-4 Adsorption ofMS2 in fractionated wastewater to soil. Notes: Samples of flushed dairy manure wastewater (influent) or anaerobically digested flushed dairy manure wastewater (effluent) were placed in dialysis tubing with molecular weight cutoffs of 100 kDa, 10 kDa, and 1 kDa. The samples were dialyzed against denionzed water for 3 days at 4C. Two ml of undialyzed (natural) or dialyzed samples were seeded with virus and mixed with 1 g of soil for 1 hr. The samples were centrifuged and the supernatant fraction was assayed to determine percent adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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112 100 D Influent 80 Effluent c::I 0 -= e60 0 ,., 'C < 40 'I20 0 Natural > lOOkDa > IOkDa > l kDa Fraction retained in wastewater Figure 6-5 Adsorption of PRD-1 in fractionated wastewater to soil. Notes: Samples of flushed dairy manure wastewater (influent) or anaerobically flushed dairy manure wastewater ( effluent) were placed in dialysis tubing with molecular weight cutoffs of 100 kDa, 10 kDa, and 1 kDa. The samples were dialyzed against denionzed water for 3 days at 4C. Two ml of undialyzed (natural) or dialyzed samples were seeded with virus and mixed with 1 g of soil for 1 hr. The samples were centrifuged and the supernatant fraction was assayed to determine percent adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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113 100 D Influent Effluent 80 i= 0 60 .:: e-i ,, 40 < 0 20 0 Natural > IOOkDa > lOkDa > l kDa Fraction retained in wastewater Figure 6-6 Adsorption of X 17 4 in fractionated wastewater to soil. Notes: Samples of flushed dairy manure wastewater (influent) or anaerobically digested flushed dairy manure wastewater (effluent) were placed in dialysis tubing with molecular weight cutoffs of 100 kDa, IO kDa, and 1 kDa. The samples were dialyzed against denionzed water for 3 days at 4C. Two ml of undialyzed (natural) or dialyzed samples were seeded with virus and mixed with 1 g of soil for 1 hr. The samples were centrifuged and the supernatant fraction was assayed to determine percent adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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114 aw OGW + < 100 kDalnf Olnf ~Inf< 100 kDa 100.0 80.0 '::le. 0 = 60.0 0 i:: c:i. .. 0 fl) "'C < 40.0 20.0 0.0 PRDl PhiX174 Figure 67 Influence of influent compounds smaller than 100 kDa on adsorption of viruses to soil. Notes: Twenty milliliters of groundwater was placed in 100 kDa MWCO dialysis tubing and dialyzed against 1 L of flushed dairy manure wastewater (influent) for 3 days at 4C. Two milliliters of groundwater (GW) influent (Inf) dialyzed GW (GW + < 100 kDa Inf) and influent used for dialysis (Inf -<100 kDa) were inoculated with viruses and mixed with 1 g of soil for 1 hr. Samples were centrifuged and the supernatant fraction was assayed to determine percent adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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i-------,---,--...,----,-----~~ 115 l.E-+-05 --1:rGW -a< 10 kDa Inf l.E-+-04 l.E-+-03 l.E-+-02 l.E-+-01 l.E+oo -+------.-----.-------.--------.------r-----r-------, 0 10 20 30 Time,min 40 50 Figure 6-8 Inactivation of MS2 by wastewater compounds smaller than IO kDa. 60 Notes: Groundwater (a) in 10 kDa MWCO dialysis tubing was dialyzed against 1 L of flushed dairy manure wastewater (influent) for 3 days at 4C MS2 was inoculated into groundwater and dialyzed groundwater ( ) Virus solutions were assayed at 10 min intervals. Each point is an average and error bars represent standard deviation (n = 3).

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= C 100 80 !60 C "' ,, < 40 20 116 r= 0.973 Xl74 Poliovirus I LSc MS2 PRDl 04--------------~----------------I 3 4 5 Isoelectric point, pl 6 Figure 6-9 Correlation between virus pl and virus adsorption to soil. 7 Notes: The isoelectric point ofMS2, PRD-1, Xl 74, and poliovirus l (LSc) was plotted against adsorption of each virus in groundwater to soil. Error bars represent the standard deviation (n = 3) of percent adsorption of each virus in groundwater to soil.

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117 Natural 00.01% Tween 80 .0.01%S0S 00.01%CTAB 100 80 20 0 MS2 PRDl PhiX174 Figure 6-10 Adsorption of viruses in groundwater with 0.01 % final concentration of detergents to soil. Notes: Two milliliters of cationic (hexadecyltrimethylammonium bromide HTAB) anionic (sodium dodecyl sulfate SOS), and nonionic (polyoxyethylene sorbitan monooleat, Tween 80) detergent solutions were seeded with viruses and mixed with 1 g of soil for 1 hr. Samples were centrifuged and the supernatant fraction was assayed to determine percentage of virus adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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118 Natural D 0.01 % Tween 80 o.01%SDS 00.0lCTAB 100 80 20 0 MS2 PRDl PhiX174 Figure 6-11 Adsorption of viruses in influent with 0.01 % final concentration of detergents to soil. Notes: Two milliliters of cationic (hexadecyltrimethylammonium bromide, HTAB), anionic (sodium dodecyl sulfate, SOS), and nonionic (polyoxyethylene sorbitan monooleat, Tween 80) detergent solutions were seeded with viruses and mixed with 1 g of soil for 1 hr. Samples were centrifuged and the supernatant fraction was assayed to determine percentage of virus adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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100 80 'I-d' 60 i 40 20 0 Natural MS2 119 00.01% Tween 80 o.01%SDS 00.01%CTAB PRDl PhiX174 Figure 6-12 Adsorption of viruses in effluent with 0.01 % final concentration of detergents to soil. Notes: Two milliliters of cationic (hexadecyltrimethylammonium bromide, HTAB), anionic (sodium dodecyl sulfate, SDS), and nonionic (polyoxyethylene sorbitan monooleat, Tween 80) detergent solutions were seeded with viruses and mixed with 1 g of soil for 1 hr. Samples were centrifuged and the supernatant fraction was assayed to determine percentage of virus adsorption. Each bar is an average and error bars represent standard devihtion (n = 3).

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120 100 Natural 00.01% Tween 80 o.0l%SDS fa0.01% CTAB 80 cf 60 C i: e-C .,, 40 20 0 MS2 PRDl PhiX174 Figure 6-13 Adsorption of viruses in groundwater to soil pretreated with detergents. Notes: Soil samples (1 g) were mixed for 1 hr with a cationic (hexadecyltrimethylammonium bromide, HTAB), anionic (sodium dodecyl sulfate, SDS) and nonionic (polyoxyethylene sorbitan monooleat, Tween 80) detergent (final concentration 0.01 %) in groundwater. Samples were washed three times with groundwater. Viruses in 2 ml groundwater were mixed with pretreated soils for 1 hr. The sample were centrifuged and the supernatant fraction was assayed to determine percentage of virus adsorption. Each bar is an average and error bars represent standard deviation (n = 3).

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121 1.2 0.8 -+--GW 0 ---Inf A 0 6 u --Eff 0.4 0.2 0 1.2 0 8 -+--GW 0 ---Inf 0 6 B u _,._Eff 0.4 0.2 0 1.2 0 8 -+--GW 0 ---Inf 0 6 C u _,._Eff 0 4 0.2 0 1.2 -+-GW 0.8 0 ---Inf 0 6 D u _...,_Eff 0.4 0 2 0 0 2 3 4 5 Time, weeks Figure 6-14. Survival of viruses in soil. Notes: MS2 (A), PRDl (B), cl>Xl 74 (C) and poliovirus 1 LSc (D) suspended in groundwater ( 0 ), influent ( 0 ) and effluent ( 0 ). Each point is an average and error bars represent standard deviation (n = 3).

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CHAPTER 7 SUMMARY OF CONCLUSIONS Anaerobic digestion has been used for treatment of various wastewaters for energy production, waste stabilization, and pathogen reduction. Anaerobic digestion at mesophilic temperatures and long HR.Ts is known to reduce the concentrations of pathogenic bacteria from various manures. Decimation during anaerobic digestion is mainly influence by operating temperature and HRT, where higher temperatures and longer HR.Ts result in higher rates of decimation. In the current study, parameters simulating the operating conditions (i.e., 28C and 3-day HRT) of the IF AS fixed-film anaerobic digester were used to determine the contribution of other factors to decimation of indicator and pathogenic bacteria. The current study investigated the influence of the indigenous micro flora in manure on the survival of indicator organisms and pathogenic bacteria in the liquid phase during fixed-film anaerobic digestion. The current study found that indicator and pathogenic bacteria were reduced by> 0.8 log10 in the presence indigenous microflora of dairy manure. The anaerobic and facultative population of the indigenous microflora were the active population contributing to decimation. Limited substrate concentration specifically affected S. aureus. In the current study, S. aureus was the only organism that could not proliferate in effluent, but was able to grow in influent. This result suggested that: 1) Staphyolococcus aureus populations could not be predicted by current indicators and 2) that the organism was limited by the lack of substrate or inhibited in the effluent. Supplementation of effluent with tryptic soy 122

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123 broth showed that inhibitory compounds were not preventing proliferation of S. aureus. This finding suggests that a necessary substrate(s) to support S. aureus was removed during anaerobic treatment of influent. Addition of a carbon source, glucose, increased the growth of S. aureus in effluent. The inability of S. aureus to proliferate in effluent may be due to the lack of sufficient carbon sources, resulting in decimation. The current results show that the indigenous microflora in manure and the lack of sufficient substrates contributes to the decimation of indigenous indicator organisms and pathogenic bacteria. These findings suggest the mechanisms of decimation of these organisms in the liquid phase during anaerobic treatment. The current study showed reductions of these organisms in the presence of indigenous micro flora at ambient temperature (28C) and a low HRT (3 days). The results of this study demonstrate the importance of indigenous micro flora in manure for sanitization of manure wastewater by fixed-film anaerobic digestion. In addition, removal of substrates necessary to support pathogenic bacteria may also play a role in decimation. Evidence presented in the current study demonstrated the role of the fixed-film in indicator and pathogenic bacteria reduction. The fixed-film may account for removal of indicator and pathogenic bacteria from the liquid phase by attachment to the biofilm. Viability, following attachment, remains to be determined However test organisms detected in the effluent were not viable, thus suggesting inactivation in the liquid phase. Earlier work presented in this study demonstrated the role of microbial competition in the liquid phase Therefore attachment to the biofilm and microbial competition may work syn e rgistically to remove and inactive indicator and pathogenic organisms during fixed film anaerobic digestion.

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124 The current study provided evidence demonstrating the adsorption and desorption behavior of selected viruses. Influent was shown to decrease viral adsorption to sandy soil. Viruses present in influent may have the potential to contaminate groundwater during land application. Land application of untreated dairy manure wastewater may increase the movement of viruses through soil, potentially contaminating groundwater. The use of untreated wastewater may cause mobilization of attached viruses through the soil matrix by interfering with adsorption mechanisms. As demonstrated with MS2, the influent may interfere with hydrophobic and electrostatic interactions between the soil and MS2 due to the increased amount of soluble organics contributed to the soil. Furthermore, influent compounds smaller than 10 kDa may cause inactivation of MS2. However, inactivation was not observed in experiments with the whole fraction of influent. Influent may also interfere with hydrophobic and electrostatic interactions between PRDl and Xl 74 to soil, where adsorption decreases. Influent compounds smaller than 100 kDa interfered with adsorption of PRD 1 and Xl 7 4 to soil. Anaerobic digestion lowers the soluble organic content and may also remove the fraction interfering with adsorption of PRDl and Xl 74, resulting in increased virus retention and decreased virus mobilization in the soil.

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BIOGRAPHICAL SKETCH Johnny A. Davis was born in Gaston, Alabama, on June 3, 1976. In 1978, he moved to Jackson, Mississippi, where he graduated from Murrah High School in 1995. He then attended Florida Agricultural and Mechanical University in Tallahassee, Florida, majoring in biology. He graduated magna cum laude with a Bachelor of Science in May 1999. In the summer of 1999, Johnny began his graduate studies at the University of Florida, under the supervision of Dr. Samuel Farrah. He completed the requirements of a master's degree in December 2001. Since then, he has been fulfilling the requirements for a Doctor of Philosophy degree under the supervision of Dr. Samuel Farrah and Dr. Ann C. Wilkie in the Soil and Water Science Department. After completion of his graduate study, he plans to pursue postdoctoral e x periences and eventually a career in academia pursing independent research interests and training the next generation of scientists. 140

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