Fixed-film anaerobic digestion : mechanisms of pathogen reduction and impacts on virus adsorption to soil

2006·4.7 MB·English

by  Davis, Johnny A

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FIXED-FILM ANAEROBIC DIGESTION: MECHANISMS OF PATHOGEN
REDUCTION AND IMPACTS ON VIRUS ADSORPTION TO SOIL
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JOHNNY A. DAVIS
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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
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Johnny A. Davis *v
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To Ethan and Joshua.
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ACKNOWLEDGMENTS
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I would like to thank Dr. Samuel R. Farrah, chair ofthe supervisory committee, for
providing support, research facilities, and guidance towards completion ofthis body of
work. I would like to extend a sincere appreciation to the co-chair ofthe supervisory
committee. Dr. Ann C. Wilkie. Herconstantpersistence and guidancepushed the work
forward to completion. Through our numerous scientific discussions and constant mental
challenging, Ihave gained atremendous appreciation formy field ofstudy and the
pursuit ofknowledge in general. I have no doubt that our interactions will help me
develop a successful career as an independent scientist. Ithank the remaining members
ofthe supervisory committee, Dr. Ben Koopman, Dr. PeterKima, and especially, Dr.
Madeline Rasche, forproviding valuable insight to career development. I thank my wife,
Nicole Davis, for herpatience and support during this longjourney. I would like to thank
myparents, Harvey Moffitt and Emma T. Davis, fortheirwisdom and encouragement. I
would also like to thank Dr. Anne Donnelly forbeing amentor and friend. I would also
liketo acknowledge David Armstrong and the DairyResearch Unit stafffor their help
during various stages ofthe 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 forproviding funding during my graduate career.
Lastly, I would like to acknowledge my reunion with mybiological parents,
Namon and Debbie Martin, and the introduction to the family I neverhad the opportunity
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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.
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TABLE OF CONTENTS
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ACKNOWLEDGMENTS
iv
LIST OF TABLES ix
LIST OF FIGURES xi
ABSTRACT
'. xii
CHAPTER
V
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 ofAnimal Manure 20
Summary 27
Purpose ofStudy 28
INDICATOR AND PATHOGENIC BACTERIA REDUCTION BY
3
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
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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 37
Growth ofBacteria in Whole and Soluble Fractions ofWastewater 38
Total aerobic, anaerobic, and facultative bacteria 38
Escherichia coli and fecal cohforms 39
Enterococcus spp 40
Salmonella spp 41
Escherichia coli 0157:H7 41
Staphylococcus aureus 41
Discussion and Conclusions 43
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 Comparabilitywith Indigenous Fecal Cohforms 57
Biofilm Attachment Studies 58
Biofilm Sampling and Examining 58
Results 59
Fecal Cohform and GFP E. coli Survival 59
Biofilm Attachment Studies 59
Discussion and Conclusions 60
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
vu
Wastewater Characteristics 69
Influence ofWhole and Soluble Fractions ofWastewater 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
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Purpose 76
Materials and Methods 77
Collection and Analysis ofSoil Samples 77
^ Collection and Analysis ofWastewater, Groundwater, and Rainwater
Samples 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 ofFlushed 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
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Risk Assessment ofViruses Following Land Application 90
Survival ofViruses in Soil 90
Discussion and Conclusions 90
7 SUMMARY OF CONCLUSIONS...^.........'...!?:..'!... 122
LIST OF REFERENCES 125
BIOGRAPHICAL SKETCH .......'. ..:... 140
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LIST OF TABLES : :, 'r
Table page
3-L Percentage ofcarbon from various sources in yeast extract 49
3-2. Characterization ofsamples used for all experiments 50
3-3. Growth ofindicator and pathogenic bacteria in the soluble fraction of
wastewater 51
3-4. Growth ofindigenous indicatorbacteria and aureus inthe whole fraction of
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wastewater 52
3-5. Comparison ofgrowth between indigenous bacteria inwastewater at 2>^°C and
28°C 53
3-6. Determination ofinhibitory ornutritional limitation on the growth ofindigenous
S. aureus 54
3-7. Growth of.S. aureus in soluble effluent and soluble effluent with various
amendments 55
5-1. Impact ofindigenous microflora on viruses suspended in groundwater and
wastewater 72
6-1. Characteristics ofviruses used in the study 97
6-2. Characteristics oftest soil 98
6-3. Adsorption ofViruses in groundwater and wastewaterto soil 99
6-4. Elution ofviruses adsorbed to soil 100
6-5. Elution ofviruses adsorbed in soil columns 101
6-6. Conductivity and pH changes in soil during batch and column experiments 102
6-7. Adsorption ofviruses in groundwater and wastewater to soil adjusted to pH 7.0. .103
6-8. Influence ofpH on adsorption ofviruses to soil 104
6-9. Elution ofviruses adsorbed to soil by detergents in groundwater 105
IX
6-10. Recovery ofviruses with 3% BE from soil pretreated with detergents 106
6-11. Estimated risk ofinfection from exposure to groundwater following land
application ofwastewater 107
X
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