• TABLE OF CONTENTS
HIDE
 Title Page
 Copyright
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Literature review
 Thiothrix antibody probe devel...
 Thiothrix sp. detection in environmental...
 Clostridium aldrichii antibody...
 Clostridium aldrichii detection...
 Summary and conclusions
 Appendix
 Reference
 Biographical sketch
 Copyright














Title: Application of antibody probes to study populations of specific bacteria in aerobic and anaerobic bioprocesses
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Title: Application of antibody probes to study populations of specific bacteria in aerobic and anaerobic bioprocesses
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Table of Contents
    Title Page
        Page i
    Copyright
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
    Table of Contents
        Page v
        Page vi
        Page vii
    List of Tables
        Page viii
    List of Figures
        Page ix
        Page x
        Page xi
        Page xii
    Abstract
        Page xiii
        Page xiv
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
    Literature review
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
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        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
    Thiothrix antibody probe development
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
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        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
    Thiothrix sp. detection in environmental systems
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
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        Page 95
        Page 96
    Clostridium aldrichii antibody probe development
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
        Page 103
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        Page 120
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        Page 122
        Page 123
    Clostridium aldrichii detection in anaerobic digesters
        Page 124
        Page 125
        Page 126
        Page 127
        Page 128
        Page 129
        Page 130
        Page 131
        Page 132
        Page 133
        Page 134
        Page 135
        Page 136
        Page 137
        Page 138
    Summary and conclusions
        Page 139
        Page 140
        Page 141
        Page 142
    Appendix
        Page 143
        Page 144
        Page 145
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    Reference
        Page 156
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    Biographical sketch
        Page 167
        Page 168
        Page 169
        Page 170
    Copyright
        Copyright 1
        Copyright 2
Full Text






















APPLICATION OF ANTIBODY PROBES TO STUDY POPULATIONS OF
SPECIFIC BACTERIA IN AEROBIC AND ANAEROBIC
BIOPROCESSES





BY

ROBIN L. BRIGMON


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


1992



























Copyright 1991

by

Robin Lewis Brigmon























To my parents
Paul and Ruby Brigmon
whose love and encouragement
made this possible









ACKNOWLEDGEMENTS

I would like to thank Dr. Gabriel Bitton, my committee

chairman, for the challenges, encouragement, and ideas that

have been part of my education. I am grateful for the

excellent technical and financial support of Dr. David

Chynoweth and Dr. Stephen Zam who made their laboratories and

resources available. My other committee members, Dr. Ben

Koopman, Dr. William McArthur, and Dr. Ammon Peck, broadened

my horizons with the different perspectives and expertise they

hold. I could call any of them for advice at any time.

Many other people were helpful and encouraging in the

completion of this work. Thanks are extended especially to

Clinton Yang for sharing his expertise in microbiology culture

techniques; my supervisors at the College of Veterinary

Medicine, Dr. Emerson Besch and Dr. Mary Christopher for their

commitment to education; Dr. Henry Aldrich and Dr. Greg Erdos

for help with the microscopy work; Gil Marshal for digester

sampling; Dr. Edward Hoffman who taught me immunology; Dr.

Ramon Littell for statistical support.

I am thankful for my mother who showed me the power of

the written word. Encouraging each other as well as exchanging

skills and talents are keys to success.

I am deeply indebted to my parents, Ruby and Paul

Brigmon, for their patience, constant encouragement and

assistance during the course of this study. I thank my wife,

Lelia, and my children, Jessica, Matthew, and Nathan, for

their patience, understanding, love, and support.

iv











TABLE OF CONTENTS





SECTION PAGE

ACKNOWLEDGEMENTS ------------------------------------- iv

LIST OF TABLES ------------------------------------- viii

LIST OF FIGURES -------------------------------------- ix

ABSTRACT --------------------------------------------- xiii

CHAPTER

1. INTRODUCTION ----------------------------------- 1

2. LITERATURE REVIEW ------------------------------- 5

2.1 Introduction ------------------------------- 5
2.2 Antibodies --------------------------------- 6
2.3 Antibody Probes in Applied Microbiology ---- 8
2.4 Activated Sludge Process -------------------- 9
2.5 Structure and Composition of Activated
Sludge Floc --------------------------------- 10
.2.6 Filamentous bacteria ------------------------ 11
2.7 Thiothrix sp. ------------------------------- 12
2.8 Filamentous Bacteria and Activated
Sludge Bulking ---------------------------- 13
2.9 Anaerobic Digestion Process ----------------- 20
2.10 Microbial Ecology of Anaerobic Digestion --- 22
2.11 Cellulolytic Clostridium sp. ---------------- 25

3. THIOTHRIX ANTIBODY PROBE DEVELOPMENT ----------- 28

3.1 Introduction ------------------------------- 28
3.2 Materials and Methods ------------------------ 29
3.2.1 Microbial Culture Procedures ----------- 29
3.2.2 Immunization Procedure ------------------- 30
3.2.3 Enzyme-Linked Immunosorbent Assay -------- 31
3.2.4 Fluorescent Antibody Procedure ---------- 32
3.2.5 Generation of Monoclonal Antibodies ------ 34
3.2.6 Specificity Assay ------------------------ 37
3.2.7 Determination of Isotype of Monoclonal
Antibodies ----------------------------- 37
3.2.8 Quantitative Absorption ----------------- 37
3.2.9 Sensitivity Assay ----------------------- 38
3.2.10 Testing for Carbohydrate Epitopes -------- 38









3.3 Results ------------------------------------ 39
3.3.1 Probe Development and Characterization --- 39
3.3.2 Purification of Monoclonal Antibodies ----- 39
3.3.3 Isotyping Monoclonal Antibodies ---------- 40
3.3.4 Quantitative Absorption Assay ------------ 41
3.3.5 Specificity for Carbohydrate Epitopes ----- 41
3.3.6 Fluorescent Microscopy -------------------- 45
3.3.7 Light and Phase Microscopy ---------------- 45
3.3.8 Sensitivity Assays ------------------------ 50
3.4 Discussion --------------------------------- 50

4. Thiothrix sp. DETECTION IN ENVIRONMENTAL SYSTEMS --- 58

4.1 Introduction ------------------------------- 58
4.2 Materials and Methods ------------------------ 59
4.2.1 Thiothrix Detection in Mixed Cultures
with ELISA ------------------------------- 61
4.2.2 Environmental Sampling Procedures -------- 61
4.2.3 Identification of Thiothrix in
Environmental Samples -------------------- 61
4.2.4 Fluorescent Antibody Procedure ---------- 62
4.2.5 ELISA of Environmental Samples ---------- 63
4.3 Results ------------------------------------ 63
4.3.1 Thiothrix Detection in Mixed Culture
with ELISA ------------------------------- 63
4.3.2 Identification of Thiothrix in
Environmental Samples -------------------- 63
4.3.3 Fluorescent Microscopy of Environmental
Samples -------------------------------- 83
4.3.4 ELISA Testing with Environmental
Samples -------------------------------- 91
4.4 Discussion --------------------------------- 92

5. Clostridium aldrichii ANTIBODY PROBE DEVELOPMENT -- 97

5.1 Introduction ------------------------------- 97
5.2 Materials and Methods ------------------------ 99
5.2.1 Microbial Culture Procedures ------------- 99
5.2.2 Immunization Procedure ------------------- 100
5.2.3 Enzyme-Linked Immunosorbent Assay -------- 101
5.2.4 Preparation of Hybridomas --------------- 102
5.2.5 Purification of Monoclonal Antibodies --- 102
5.2.6 Determination of Isotype of Monoclonal
Antibodies ------------------------------- 104
5.2.7 Quantitative Absorption ------------------ 104
5.2.8 Testing for Carbohydrate Epitopes -------- 105
5.2.9 P1 Digester Isolate Assays --------------- 105
5.2.10 Cross-reactivity Assay ------------------- 106
5.2.11 Sensitivity Assay --------------------- 106
5.2.12 Ultra-immunocytochemistry ---------------- 106
5.3 Results ------------------------------------ 107
5.3.1 Specificity Assays ----------------------- 107
5.3.2 Sensitivity Assays ----------------------- 109
5.3.3 Isotype of Monoclonal Antibodies --------- 113









5.3.4 Specificity for Carbohydrate Epitopes -----114
5.3.5 Quantitative Absorption Assay ------------- 114
5.3.6 Immunocytochemical Testing --------------- 118
5.4 Discussion ---------------------------------- 118

6. Clostridium aldrichii DETECTION IN ANAEROBIC
DIGESTERS --------------------------------------- 124

6.1 Introduction ------------------------------- 124
6.2 Materials and Methods ---------------------- 125
6.2.1 Clostridium aldrichii Detection in Mixed
Culture with ELISA ---------------------- 125
6.2.2 Analysis of Anaerobic Digester Effluent
with addition of Clostridium aldrichii --- 125
6.2.3 Anaerobic Digesters --------------------- 126
6.2.4 Anaerobic Digester Sampling -------------- 126
6.2.5 ELISA of Anaerobic Digester Effluent ------ 126
6.2.6 Immunocytochemical Examination of S12
Samples using PAB ------------------------- 128
6.3 Results ------------------------------------ 128
6.3.1 Clostridium aldrichii Detection in Mixed
Culture with ELISA ---------------------- 128
6.3.2 Analysis of Anaerobic Digester Effluent --- 128
6.3.3 Results of Anaerobic Digester Testing ----- 133
6.3.4 Immunocytochemical Results ---------------- 133
6.4 Discussion ---------------------------------- 135
7. SUMMARY AND CONCLUSIONS --------------------------- 139


APPENDICES

A. PROCEDURES FOR THE GRAM STAIN, INTRACELLULAR SULFUR
GRANULE TEST, AND SHEATH STAIN -------------------- 143

B. WATER CHEMISTRY DATA FROM ENVIRONMENTAL
SAMPLING SITES ----------------------------------- 145

C. REAGENTS USED IN IMMUNOASSAYS ------------------- 148

D. REAGENTS USED IN HYBRIDOMA PRODUCTION ----------- 150

E. BACTERIAL GROWTH MEDIUMS ------------------------- 151

REFERENCES --------------------------------------- 156

BIOGRAPHICAL SKETCH ----------------------------------- 167












LIST OF TABLES


Table Page

2-1 Type of compaction and settling interference caused
by various filamentous organisms ------------------- 14

2-2 Dominant filament types as indicators of conditions
causing activated sludge bulking ------------------- 19

2-3 Microbial synergism in the digestion of hemicellulose
and pectin ------------------------------------------ 23

3-1 ELISA screening of Thiothrix sp. monoclonal
antibodies to select bacteria ----------------------- 40

3-2 ELISA isotype testing of monoclonal antibodies
developed against Thiothrix sp. --------------------- 42

3-3 Quantitative absorption of specific monoclonal
antibodies with ELISA ------------------------------- 43

3-4 Effect of periodate oxidation of Thiothrix Al on the
binding of MABs 16010, 3511, and 9168 ------------- 44

4-1 ELISA testing for the presence of Thiothrix in
environmental samples using monoclonal antibodies --- 92

5-1 Specificity of ELISA for P1 isolates using
different antibody concentrations ------------------- 108

5-2 ELISA screening of Clostridium aldrichii specific
monoclonal antibodies to select bacteria ---------- 110

5-3 ELISA isotype testing of monoclonal antibodies
developed against Clostridium aldrichii ------------ 115

5-4 Effect of periodate oxidation of Clostridium
aldrichii on the binding of MABs 57 and 94 --------- 116

5-5 Quantitative absorption of specific monoclonal
antibodies with ELISA ------------------------------- 117

6-1 Anaerobic digester characteristics ----------------- 127

6-2 ELISA of Poplar-fed S-12 digester effluent --------- 132

6-3 ELISA testing of wood-poplar anaerobic digesters S12
and S5 effluent for Clostridium aldrichii ---------- 134


viii












LIST OF FIGURES


Figure Page

2-1 Schematic representation of the production of
polyclonal and monoclonal antibodies --------------- 7

2-2 Categories of metabolically distinct bacteria in
the methane fermentation --------------------------- 21

3-1 Schematic of the ELISA protocol -------------------- 33

3-2 Schematic of the FAB protocol ---------------------- 35

3-3 Fluorescent immunoassay testing Thiothrix type Al
with MAB 3511 at 400X ------------------------------ 46

3-4 Fluorescent immunoassay testing Thiothrix type Q
with MAB 3511 at 400X ------------------------------ 47

3-5 Leucothrix type N2 with bright field microscopy at
400X ----------------------------------------------- 48

3-6 Type "021N" 400X treated with modified Gram stain -- 49

3-7 Thiothrix type 3 with sheath stain at 400X --------- 51

3-8 Thiothrix type Q treated with modified Gram stain
400X ----------------------------------------------- 52

3-9 Relationship between absorbance and concentration of
Thiothrix Al protein using ELISA with clone 9168 --- 53

3-10 Relationship between absorbance and concentration of
Thiothrix Al protein using ELISA with clone 3511 --- 54

3-11 Relationship between absorbance and concentration of
Thiothrix Al protein using the ELISA with PAB ------ 55

4-1 Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with PAB
in 10 % sludge (w/v) ------------------------------ 64

4-2 Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with MAB
3511 in 10 % sludge (w/v) ------------------------- 65

4-3 Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with MAB
9168 in 10 % sludge (w/v) ------------------------- 66









4-4 Thiothrix sp. in sulfide enriched activated sludge
sample from the UF plant 400X ---------------------- 68

4-5 Thiothrix sp. in LTH with WMS water enriched activated
sludge sample from the Main Street plant 400X showing
signs of "cross-bridging floc" particles ---------- 69

4-6 Thiothrix sp. in LTH with WMS water enriched activated
sludge sample from the Main Street plant 400X showing
signs of "twitching" or swaying motility ---------- 70

4-7 Thiothrix nivea rosette form Warm Mineral Springs
at 1000X ------------------------------------------- 72

4-8 Thiothrix nivea from Warm Mineral Springs with sheath
stain at 400X ------------------------------------- 73

4-9 Thiothrix nivea next to filaments of Beqqiatoa
gigantea from Warm Mineral Springs at 100X --------- 74

4-10 Thiothrix nivea growth or "bloom" in City of Palatka
water storage tank aeration system ----------------- 75

4-11 Thiothrix nivea darkfield micrograph (100X) of fresh
sample from City of Palatka water storage tank
aeration system ------------------------------------ 76

4-12 Thiothrix nivea darkfield micrograph (100X) of fresh
sample from City of Palatka water storage tank
aeration system with different lighting ----------- 77

4-13 Thiothrix nivea attached to algae in darkfield
micrograph (400X) of fresh sample from City of Palatka
water storage tank --------------------------------- 79

4-14 Thiothrix sp. at sulfur seeps in Florida Orange
Spring at a depth of approximately 40 feet --------- 81

4-15 Thiothrix sp. in spring run at Florida Orange Spring
attached to Charas (Phase Contrast, 400X) ---------- 82

4-16 Thiothrix nivea attached to diatoms in phase contrast
micrograph (400X) in fresh sample from Warm Mineral
Spring --------------------------------------------- 80

4-17 Thiothrix sp. in pipe which caused irrigation system
blockage and subsequent crop damage in Polk County,
Florida. SEM by Dr. Henry Aldrich, Department of
Cell Science and Microbiology ---------------------- 84

4-18 Main street plant sample of activated sludge with
numerous filamentous bacteria seen at 100X on light
microscopy --------------------------------------- 85










4-19 Main street plant sample of activated sludge with
numerous filamentous bacteria seen at 100X with
fluorescent microscopy having been treated with the
fluorescent immunoassay using MAB 3511 ------------- 86

4-20 Main street plant sample of activated sludge with
numerous filamentous bacteria seen at 100X with
fluorescent microscopy having been treated with the
fluorescent immunoassay using MAB 9168 ------------- 87

4-21 Phase-contrast micrograph of sample from Orange Spring
treated with the fluorescent immunoassay using MAB
3511 (100X) --------------------------------------- 88

4-22 Fluorescent micrograph of sample from Orange Spring
treated with the fluorescent immunoassay using MAB
3511 (100X) --------------------------------------- 89

4-23 Fluorescent micrograph of sample from Warm Mineral
Spring treated with the fluorescent immunoassay using
MAB 3511 (100X) ----------------------------------- 90

5-1 Relationship between absorbance and concentration
of C. aldrichii using the ELISA with PAB ---------- 111

5-2 Relationship between absorbance and concentration
of C. aldrichii using the ELISA with MAB 94 -------- 112

5-3 Relationship between absorbance and concentration
of C. aldrichii using the ELISA with MAB 57 -------- 113

5-4 Electron microscopy of Clostridium aldrichii (PI) cells
(a) Thin section treated with PAB and IgG-gold complex;
(b) Control experiment performed by treating a section
with pre-immune plasma and IgG-gold complex ------- 119

5-5 Thin sections treated with PAB and IgG gold complex. (a)
Clostridium populeti cells. (b) Fibrobacter
succinogenes. (c) Methanosaetii concilii.
(d) Streptococcus mutans. --------------------- 120

6-1 Relationship between absorbance and concentration
of C. aldrichii using the ELISA with PAB
in 10 % sludge (w/v) ------------------------------- 129

6-2 Relationship between absorbance and concentration
of C. aldrichii using the ELISA with MAB 94
in 10 % sludge (w/v) ------------------------------- 130









6-3 Relationship between absorbance and concentration
of C. aldrichii using the ELISA with MAB 57
in 10 % sludge (w/v) ------------------------------- 131

6-4 S-12 anaerobic digester sample. Thin section treated
with PABs and IgG-gold complex -------------------- 135


xii














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


APPLICATION OF ANTIBODY PROBES TO STUDY POPULATIONS
OF SPECIFIC BACTERIA IN AEROBIC AND
ANAEROBIC BIOPROCESSES


by

Robin L. Brigmon

August 1992

Chairman: Gabriel Bitton
Cochairman: David P. Chynoweth
Major Department: Environmental Engineering Sciences


Immunological research with microorganisms is commonplace

today. Current advances in biotechnology have lead to

sensitive, accurate, and reproducible procedures for bacterial

detection. However, the focus has been primarily on pathogenic

microorganisms. Other than methanogenic and plant-associated

bacteria, little attention has been focused on important

bacteria in the environment.

In recent years, there has been an increasing concern with

respect to the preservation of the environment. Conservation

of land use, water and energy are of major concerns. The use

of microorganisms for waste treatment is popular because of

its efficiency, flexibility and low space requirements as well

as being a natural process. One aspect that needs

clarification, and is still incompletely understood, is the


xiii









composition of the microbial consortia that converts waste

into useful or harmless molecules.

It is clear that in order to maximize efficiency of these

systems a better understanding of the microbes in the

bioprocess is essential.

This work was designed (1) to develop antibody probes as

tools to detect and quantify specific bacteria in complex

biological systems, (2) to compare these probes to standard

microbiological techniques used to detect these species and

(3) to determine methodologies for best use of these probes in

actual bioprocess monitoring. Monoclonal and polyclonal

antibodies were developed for a filamentous aerobic sulfur

bacterium, Thiothrix sp., and a cellulolytic anaerobe,

Clostridium aldrichii. Both bacteria are important in

biological treatment processes.

Thiothrix sp. were detected in two activated sludge

systems at the University of Florida and the city of

Gainesville, Florida, a municipal water storage tank in

Palatka, Florida, irrigation pipe samples from central

Florida, and two Florida springs. Clostridium

aldrichii was monitored in anaerobic digesters maintained in

the Department of Agricultural Engineering on the University

of Florida campus over two years. The immunoassays developed

were rapid and proved highly accurate. Antibody probes,

particularly monoclonal antibodies, are potentially useful as

biosensors in monitoring natural and engineered ecosystems.


xiv















CHAPTER 1
INTRODUCTION




Design and operation of man-made biological systems is

currently based largely on empirical data and little knowledge

of the specific groups of bacteria involved. Indicator

organisms are utilized in procedures as a measure of the

efficiency of a specific system (Brock and Madigan, 1991). The

use of indicator organisms is often based on their ease of

isolation and culture (APHA, 1989). Lack of information on

specific bacteria can be attributed to the difficulty,

expense, and extensive manpower effort required for their

isolation and identification. This can take as much as 60 days

for some species (Wedekind et al., 1988). The need is

therefore apparent for tools which permit the rapid

characterization of specific bacteria.

Recent advances in biotechnology have provided a number of

such tools in addition to making them economically feasible

(Brigmon et al., 1992, Peck and Archer, 1991, Macario and de

Macario, 1985). The goal of the proposed research is to use

antibody probes to study specific bacterial populations in

biological treatment processes. These antibodies were

developed and first tested in the laboratory for calibration

1








2

purposes. Antibody probes were then used as tools in testing

actual processes in which they would be used as tests for

specific populations. These techniques involve detection via

specific antibody probes which would be in turn detected by a

secondary labeled probe.

These immunological techniques were applied to Thiothrix

type Al (TX), a filamentous aerobic sulfur bacterium and

Clostridium aldrichii (P1), a cellulolytic anaerobe, from two

distinct biotreatment processes. The anaerobic processes

tested were anaerobic digesters operating on the University

of Florida campus. Aerobic processes tested included primarily

activated sludge samples obtained locally. Previous work has

shown these two microorganisms to be significant in terms of

their impact on the microbial ecology of their respective

systems.

The activated sludge process is extremely important in

waste treatment in terms of public health and pollution

control because of its efficiency, flexibility, and low space

requirements. Bulking, or swelling of the sludge in volume

which can impair plant operations, usually is caused by

excessive growth of filamentous organisms. Thiothrix sp. are

one of these problem causing microbes (Farquhar and Boyle,

1972, and Jenkins et al., 1984). Overgrowth of Thiothrix sp.

in irrigation systems is costly in terms of repair of blocked

pipeline and emitters, crop loss, as well as wasting water

(Ford and Tucker, 1975, and Pitts et al., 1990)








3

Anaerobic digestion was originally used for treatment of

sewage sludges and more recently is being developed for

conversion of energy crops and municipal, agricultural,

industrial wastes to methane gas. Imbalances frequently occur

in these systems which result in inhibition of the overall

conversion to methane. Such imbalances would be better

understood through knowledge of the specific organisms

involved and their population and activity dynamics. C.

aldrichii has been repeatedly isolated from the same wood-fed

digester over a period of two years making it a key microbe

in this system (Yang et al., 1990).

My research with these two species is designed to (1)

develop antibody probes specifically for these two bacteria,

Thiothrix Al and Clostridium aldrichii (2) test immunoassay

protocols for use in detection and quantification of test

organisms, and (3) compare immunological detection methods

with traditional microbiological procedures with actual

samples from these diverse bioprocesses.

The overall objective of the proposed research was to

study specific bacteria in complex biological systems.

Research will focus primarily on development of immunological

techniques for the identification, study, and quantification

of select microorganisms. I have chosen as my models for this

study an aerobic and an anaerobic bacteria. Emphasis was

placed in the area of process control.

More specifically, the objectives of this study are as










follows:

1. Development of monoclonal antibodies (MABs) and

polyclonal antibodies (PABs) specific for selected

microorganisms Thiothrix type "Al" (Tx) and Clostridium

aldrichii (P1). Antibody development required working with

mice, bacteria and mammalian cell cultures, screening of

antibody preparations for specificity, and calibration of

antibodies for sensitivity.

2. Testing antibodies in different assay systems

including enzyme-linked immunosorbent assay (ELISA),

fluorescent-antibodies (FAB), and electron microscopy (EM).

3. Immunological detection of P1 and Tx in their

bioprocess environment. Test for P1 in anaerobic digesters.

Test for Tx in aerobic sludge, spring, and water

treatment system samples.

4. Study systems over time to test for correlation of

specific populations with present bioprocess indicators.

This task required sampling of digesters overtime and

comparing P1 detection with methane data. At the same time

monitoring Tx populations and following treatment processes.

5. Compare culture and morphology identification

techniques with 2. and 3..










CHAPTER 2
LITERATURE REVIEW



2.1 Introduction

Animals possess an extremely advanced mechanism, the

immune response, for developing resistance or protection to

specific microorganisms. Two systems constitute the vertebrate

immune response: the cellular and humoral systems (Clark,

1983). These are based on two types of lymphocytes, the T-

cells and B-cells, respectively. When a specific B-cell of the

humoral system recognizes and interacts with a complementary

specific foreign entity (referred to as an immunogen), it is

activated to differentiate into a plasma cell which then

undergoes multiplication and forms clones of the original

plasma cell. The resulting plasma cells synthesize and secrete

molecules of a specific glycoprotein (the antibody or

immunoglobin) into the blood plasma. The immune response is a

defense mechanism for the neutralization of foreign

macromolecules including protein, nucleic acids, or

polysaccharides, which operates in part through the formation

of specific antibodies. Invading bacteria contain a variety of

macromolecules foreign to the host, and antibodies develop

against it. Only specific localized areas on the molecular

surface, called epitopes, actually interact with complementary

surface receptors on the B-cell to initiate the immune

response.










2.2 Antibodies

The proteins known as antibodies or immunoglobins have

played a critical role in research, medicine and biotechnology

since the beginning of modern biochemistry (Edelman, 1970).

Their key function in the immune system has made these

molecules and their part in the body's disease-fighting

mechanisms the subject of intensive study. Immunoglobins

react with antigens through noncovalent interactions of

particular amino acids in the antigen combining site, with

various macromolecules on the surface of the antigen called

antigenic determinants (Clark, 1983). There is nothing

mysterious about this particular reaction since it is governed

by the same laws of thermodynamics as any other chemical

reaction. Antibodies have almost unbelievable diversity and

extreme specificity of interaction with their corresponding

antigens. These qualities have made them very powerful tools

in the hands of the biochemist and clinician for the

detection, assay, and visualization of other biomolecules .

Each single, specific antibody is produced by a plasma

cell clone derived from a single B-cell stimulated by a single

complementary epitope. Such a single, specific antibody is

called a monoclonal antibody. Because a given immunogen such

as a bacterium can have many epitopes, each with a

corresponding plasma cell clones and complementary antibodies,

a whole serum derived from a single immunogen is a

monospecific polyclonal antiserum. Figure 2-1 shows a











2 3 Antigenic determinant

Immunogen


Spleen


B-Lymphocytes B-Lymph
@@ o @e

~I-








Polyclonal Antiserum I



Mixture of antibodies M


Figure 2-1


ocytes


Myeloma cells


@@ 00

Fusion


00


hybrid myeloma cells
) @ >
I I I
Clones





monoclonal antibodies

monoclonal antibodies


Schematic representation of the production of
polyclonal and monoclonal antibodies (From
Oellerich, 1983)








8

schematic representation of the production of polyclonal and

monoclonal antibodies. With the advent of monoclonal

antibodies (Kohler and Milstein, 1975) and methods of large

scale production, the widespread use of antibodies in therapy

and laboratory or production-scale purification has also

become possible (Zimmerman et al., 1985).

2.3 Antibody probes in Applied Microbiology

Currently, the term "antibody probe" has been used to

include both monoclonal antibodies (MABs) and polyclonal

antibodies (PABs) used in various immunological procedures

proven to be specific for the molecule or cell for which they

were developed (Kobayashi et al., 1988). Immunologic

detection of microorganisms, for example, does not

necessarily require isolation and culture of specific

organisms. Comparisons of both methods, immunologic and

isolation, have proven favorable in environmental studies of

specific bacterial populations (Brigmon et al., 1992, Cherry

et al., 1972).

Fluorescent antibodies (FAB) may be used for direct

observation of microorganisms. This technique involves

staining the microorganism with a fluorescent dye that is

coupled to an antibody. Visualization of the staining reaction

is by means of fluorescent microscopy. A number of bacteria,

including ecologically important organisms involved in key

geobiochemical cycling reactions, have been examined in their

natural habitats. Rhizobium japonicum has been followed in the








9

soil in association with specific plant roots in the presence

of other Rhizobium species (Bohlool and Schmidt, 1973).

Immunological methods that have been used to study

methanogenic populations in anaerobic digesters include

enzyme-linked immunosorbent assays (ELISA) (Kemp et al., 1988;

Gorris, 1987), electron microscopy (EM) (Robinson and Erdos,

1985), and fluorescent antibodies (FABs) (De Macario et al.,

1982). In recent years there has been increased interest in

these bacteria because of their role in degradation of organic

wastes from agricultural and food industries to reduce

pollution and to produce methane as fuel (Macario et al.,

1989). Anaerobic bacteria, particularly the methanogens, tend

to grow very slowly and are extremely fastidious so these

techniques are of great value for their study.

2.4 Activated Sludge Process

The activated sludge process of wastewater treatment is a

biological contact-process first developed in England by Arden

and Lockett (1914). The term comes from the active consortium

of microorganisms including bacteria and protozoans which

process materials originate in the sewage itself aerobically

stabilizing the material (Imhoff and Fair, 1940). The solids

and organic present in the sewage form the initial matrix and

substrate for biological growth and metabolism. Subsequent

development depends largely on the waste materials dispersed

in the sewage by mechanical mixing and aeration increasing

biological contact. Large flocs of suspended matter are formed








10

by attachment of particulate waste materials and

microbiological growth. These flocs are covered with

biological activity and by the constant feeding, cleanse or

restore the active contact surface, hence the term "activated"

sludge flocs.

If flocs are permitted to settle and then added to fresh

sewage or wastewater that is again aerated, then flocculation

occurs in a shorter time period. The sedimented floc thus

developed for addition to fresh sewage is designated

"activated sludge." The activated sludge process is one of the

most important methods for sewage treatment.

2.5 Structure and composition of activated sludge floc.

Floc particles are gelatinous in nature and contain a

large number of bacteria, many of which form polysaccharides.

Slime-forming bacteria, Zooqloea sp. (Farrah and Unz, 1976),

as well as materials in incoming wastewater and extracellular

polymers (Pavoni et al., 1972) can combine to form flocs.

These flocs form the substratum to which a mixed consortium of

protozoa, fungi, yeasts, algae, other bacteria, and inert

particles in the wastewater can then attach or be enmeshed in

the matrix. As the system is aerated and mixed with fresh

sewage, the floc particles absorb suspended and colloidal

matter, increase in size, and break into smaller units that

repeat the same process. These flocs are supplied with a

continuous supply of air, providing ideal conditions for

oxidation.










2.6 Filamentous bacteria.

There is a large variety of filamentous bacteria present

in the environment (Brock and Madigan, 1991). Green bacteria

are morphologically diverse and include the motile filamentous

gliding species Chloroflexus, Heliothrix, and Oscillatoria

which are anoxygenic phototrophs. The cyanobacteria comprise

a large and heterogeneous group of oxygenic phototrophic

organisms including the filamentous Oscillatoria and Anabena.

Sheathed bacteria are filamentous organisms with a unique

life cycle involving formation of flagellated swarm cells

within a long tube or sheath. Sheathed bacteria such as

Sphaerotilus, Leptothrix, and Crenothrix species are common in

flowing freshwater habitats that are rich in organic matter

such as polluted streams, trickling filters, and activated

sludge systems (APHA, 1989). Sphaerotilus has often been

referred to as the "sewage fungus" due to its filamentous

appearance in association with polluted water.

The actinomycetes are a large group of filamentous

bacteria which form branching filaments which include the

species Actinomycetes, Nocardia, and Streptomyces. Nocardia

species are frequently associated with foam formation in the

activated sludge process (Goddard and Forster, 1991).

Lithotrophic bacteria are physiologically united by their

ability to utilize inorganic electron donors as energy sources

including the filamentous sulfur lithotrophs Beggiatoa and

Thiothrix species (Larkin and Strohl, 1983).










2.7 Thiothrix sp.

The first description of the organism we know as Thiothrix

was made in 1865 by Rabenhorst who named the organism

Beqqiatoa nivea. Winogradsky transferred it to his newly

created genus Thiothrix as the type species in 1888. The genus

was created for organisms that (a) deposit sulfur internally

when in the presence of sulfide, (b) produce ensheathed

filaments that may attach to substrates, (c) produce gliding

gonidia from the unattached end of the filament, and (d)

produce rosettes (Larkin & Shinabarger, 1983). However, due to

difficulties in obtaining pure cultures, (Harold and Stainer,

1955, Caldwell et al., 1975, Bland and Staley, 1978), it was

only in recent years that Thiothrix sp. have been well

characterized (Larkin, 1980, Williams and Unz, 1985).

The ecology and distribution of Thiothrix and other

sulfur-oxidizing bacteria has been reviewed by Lackey et al.

(1965) and Larkin and Strohl (1983). Thiothrix sp. have the

ability to fix nitrogen in aquatic environments (Polman and

Larkin, 1990). The primary natural habitat of Thiothrix is

sulfide-containing flowing water (Larkin & Shinabarger, 1983)

where it's ability to attach to objects in sulfide-rich waters

gives it an advantage over nonperiphytic bacteria. They have

been reported in a wide range of environments from cool (100C)

(Bland and Staley, 1978) and warm (310C) (Lackey et al., 1965)

springs.








13

2.8 Filamentous bacteria and activated sludge bulking.

Thiothrix sp. have been observed in the biomass of aerobic

wastewater treatment systems (Williams and Unz, 1985) and

caused blockage problems in drip irrigation systems (Ford and

Tucker, 1975). In 1939, Thiothrix was observed in association

with Beqqiatoa and Sphaerotilus in growth of waste water fungi

(Cook, 1954) and was observed in activated sludge samples

taken from the Hiroshima City, Japan wastewater treatment

plant (Lackey et al., 1965). Thiothrix sp. were observed in 12

of 16 activated sludge samples examined in 1971 (Farquhar and

Boyle, 1971b). They were the dominant form in six samples,

five of which were in a bulking sludge. At all five of these

sites there existed a smell of H2S.

Bulking is a macrostructure problem in which the

filamentous bacteria that provide the macrostructure

predominate in the system (Jenkins et al., 1984). There are a

number of filamentous bacteria that can cause bulking. The

overgrowth of these filamentous bacteria interferes with

compaction and settling of the activated sludge either by

producing a very diffuse floc or by over growth in the mixed

liquor and causing bridging between flocs. The type of bulking

problem depends on the causative filamentous bacteria. Table

2-1 indicates types of interference caused by the most common

filamentous organisms observed to cause compaction and

interference problems in activated sludge. There are early

reports linking activated sludge bulking to the presence of














Table 2-1


Type of compaction and settling interference
caused by various filamentous organisms


Bridging


Open Floc Structure


type 021N

Sphaerotilus natans

type 0961

type 0803

Thiothrix sp.

type 0041

Haliscomenbacter hydrossis


type 1701

type 0041

Microthrix parvicella

type 0675

Nostocoida limicola


(From Jenkins et al., 1984)








15

H2S without the mention of Thiothrix sp. as the causative

microorganism (Greeley, 1945). It has been observed that H2S

concentrations in the range of 0.3 mg/l to 0.5 mg/l caused

activated sludge to bulk. Some have described filamentous

bacteria which bear a close resemblance to Thiothrix in

association with bulking but could not identify the organism

(Hatfield, 1931). Excessive growth and development of these

bacteria may confer on the normal flocculent activated sludges

in sewage treatment plants poor settling and compaction

properties, resulting in a condition known as filamentous

bulking (Jenkins et al., 1984).

Thiothrix sp. have frequently been associated with

activated sludge bulking (Farquhar and Boyle, 1972), a problem

in wastewater treatment systems. This interferes with the

waste treatment process and can cause economic as well as

public health problems. Bulking, or the swelling of the sludge

in volume, is associated with the loss of sludge activity.

Sludge activity is the rate at which oxygen is used up by the

sludge in the presence of fresh sewage. True bulking is

characterized by a poor settling rate and a high sludge volume

index (SVI). SVI is obtained by taking 1 liter of well-mixed

fresh activated sample in a 1 liter graduate cylinder and

letting it settle for 30 min. A reading is then taken based on

the volume of settled sludge relative to the mixed liquor

suspended solids (MLSS) which gives the SVI (ml/g). Bulking

sludge would have an SVI in excess of 150 ml/g.








16

Detailed knowledge of the ecology and taxonomy of

wastewater filamentous sulfur bacteria has been hampered due

to the difficulty of obtaining axenic strains for comparative

study (Wanner et al., 1987). Larkin obtained the first pure

culture of T. nivea in 1980 (Larkin, 1980). Growth of this

isolate required a reduced sulfur source, an organic carbon

source (acetate, malate, pyruvate, and oxalacetate served as

sole carbon sources), and C2O, thereby establishing the

mixotrophic mode of nutrition for T. nivea (Larkin &

Shinabarger, 1983 and Strohl & Schmidt, 1984). Recent

developments in culture techniques have indicated that T.

nivea from a Florida Spring is best cultured when using the

water in media from where it was originally isolated

(McGlannan and Makemson, 1990). The development of Thiothrix

populations in aquatic environments may provide a useful

indicator of sulfide contamination, while at the same time

contribute to the detoxification of the affected system

without imposing a significant oxygen demand (Jones, et al.,

1982).

Decreasing aeration in activated sludge systems have been

found to have a positive effect on suppressing filamentous

organisms such as Sphaerotilus natans and Type 021N. However,

this decrease in aeration has caused filamentous bulking by

Thiothrix sp. (Wanner et al., 1987). Preaeration of wastewater

was found to have a positive effect of reducing activated

sludge bulking by removal of sulfide (Farquhar and Boyle,








17

1972). Thiothrix sp. utilize sulfur as an energy source

(Schmidt et al. 1987) and forms sulfur inclusion bodies

(Williams et al. 1987). Therefore, sulfide was found to be the

limiting factor for the growth of Thiothrix sp. in the sludge.

Chlorination has been successful in the control of

Thiothrix sp. related problems in 4 wastewater treatment

plants (Jenkins et al., 1984). In irrigation systems chlorine

has been effectively used for Thiothrix sp. control, although

it has to be continually applied to prevent build-up in pipes,

valves, or emitters (Ford and Tucker, 1975, Ford, 1979a).

The application of ferrous sulfate in preventing or

controlling bulking sludge problems caused by Thiothrix sp.

has proven successful in the laboratory (Lee, 1987). Different

doses of ferrous sulfate were required for bulking caused by

Thiothrix vs Sphaerotilus natans. Thus, identification of

etiological agent is important for correct treatment.

A manual was published to help deal with the problem of

activated sludge bulking and foaming as it has proven to be a

common problem in activated sludge plants throughout the world

(Jenkins et al. 1984). A description of known organisms

causing filamentous bulking problems under various conditions

is presented in the manual, recommendations are given to

remedy the difficulty. It is presently difficult to

positively differentiate these microorganisms which can make

a vast difference in terms of treatment. Table 2-2 summarizes

specific cause and effects that have been established for a










few filamentous organisms.

Thiothrix sp. have also been observed in biological foams

in activated sludge plants (Jenkins et al., 1986). Stable

brown viscous foams form a layer which can be detrimental to

the operation of the plant. Technical solutions for foaming

include addition of oxidants, anti-foaming agents, coagulant

addition, mechanical means, and change in operation (Pujol et

al., 1991). Removal can be complicated, costing time and

money. The presence of Thiothrix sp. in some samples can be

related to a nutritional imbalance in the foam medium which

can remain on the surface of treatment tanks for a long time.

For the case of foaming (Pujol et al., 1991) as in bulking

(Faraquhar and Boyle, 1972), precise identification of the

bacteria involved, together with a thorough knowledge of their

physiology, will remain the basis for selecting and applying

reliable solutions allowing the control of the growth of all

filamentous microorganisms.

A recent bulletin offered advice on methods to prevent

emitter plugging in micro irrigation systems which can be

caused by what has been termed "sulfur slime" which is

produced by filamentous bacteria that can oxidize hydrogen

sulfide and produce insoluble elemental sulfur (Pitts et al.,

1990). Numerous drip irrigation systems have been installed in

central and south Florida agricultural areas (Ford & Tucker,

1974). Many have ceased to function within two weeks due to

filter and emitter blockage with "sulfur slime" (Ford, 1979b).













Table 2-2 Dominant filamentt types as indicators of conditions
causing activated sludge bulking


Suggested Causative Conditions


Low DO


Low F/M


Septic Wastewater/Sulfide


Nutrient Deficiency




Low pH


Indicative Filament Types


type 1701, S. natans, H.
hydrossis

Microthrix parvicella, I.
hydrossis, Nocardia sp.
types 021N, 0041, 0675,
0092, 0581, 0961, 0803

Thiothrix sp., Begqiatoa
and type 021N

Thiothrix sp., S. natans
type 021N, and possibly
H. hydossis and types
0041 and 0675

fungi


(From Jenkins et al., 1984)








20

2.9 Anaerobic Digestion Process

Microbial production of methane is a natural process

occurring in anaerobic environments such as ocean and lake

sediments, marshy soils, digestive tracts of insects and

larger animals (Chynoweth, 1989). This process is not only

critical to the cycling of carbon in nature but also has been

harnessed into a process called anaerobic digestion for

conversion of renewable resources to produce useful methane

gas. The rise of ambient atmospheric levels of methane

resulting from this process is also of concern to atmospheric

scientists (Peltier and Tushingham, 1989). In addition,

metabolism of methane bacteria is important to digestion,

nutrition, as well as milk and meat production by ruminants

(Hungate, 1966).

Methanogenic decomposition is a process which only occurs

under strict anaerobic conditions involving the concerted

action of many bacteria to decompose organic matter to carbon

dioxide and methane, as illustrated in Figure 2-2. The first

step carried out by nonmethanogenic bacteria involves

hydrolysis of high-molecular-weight, polymeric compounds into

their component units (Sleat and Mah, 1987). After hydrolysis

acid-forming bacteria ferment the subunits to a group of

extracellular intermediates including acetate, hydrogen, and

carbon dioxide. These intermediates are converted to methane

by a fastidious, slow-growing population of methanogenic

bacteria. A balanced relationship between these two diverse






















COMPLEX
ORGANIC
CARBON


TRANSITIONAL
BACTERIA


I HYDROLYTIC
BACTERIA

ORGANIC ACIDS
NEUTRAL COMPOUNDS


II
HYDROGEN PRODUCING
ACETOGENIC BACTERIA


III
HOMOACETOGENIC
H2/COz BACTERIA
ONE CARBON ------------ ACETIC ACID
COMPOUNDS I
III 1


METHANOGENIC
BACTERIA


CH4 + CO2


Figure 2-2. Categories of metabolically distinct bacteria in
the methane (From Chynoweth, 1989)








22

groups of bacteria is needed for a stable high performance

fermentation.

2.10 Microbial Ecology of anaerobic digestion

The rate-limiting step in the degradation of cellulose

during continuous culture of Ruminococcus albus and

Methanobrevibacter smithii was observed to be cellulose

hydrolysis. The coculture produced more H2 and. acetate but

less ethanol than a mono-culture of R. albus, indicating

interspecies H2 transfer was operative during coculture

(Pavlosthasis, 1990).

Even with animals fed diets containing cellulose as the

sole source of carbohydrate, nearly two thirds of the bacteria

which can be isolated on non-selective media are non-

cellulolytic. A consortium of bacteria attach and degrade

plant material in the rumen (Akin and Barton, 1983). Some of

the cellulose degrading bacteria have enzymes capable of

degrading hemicellulose and pectin but many strains make

little or no use of the degradation products for growth. Their

involvement in degrading these compounds and releasing soluble

material for other microbes to ferment is probably as

important to the function of the bioprocess as their

cellulolytic activity. An example of microbial

interactions which can occur between cellulolytic and non-

cellulolytic species during the degradation of cellulose and

pectin by Ruminococcus flavefaciens and Bacteroides ruminicola

can be seen in Table 2-3.












TABLE 2-3. Microbial synergism in the digestion of
hemicellulose and pectin.




Brome grass (boot) Brome grass (bloom)


Degradation Fermentation Degradation Fermentation
(%) (%) (%) (%)

Hemicellulose

B. ruminicola 4.7 6.1 5.0 6.1


R. flavefaciens 77.8 0.0 61.1 0.0

Mixed culture 84.1 80.3 70.3 67.2

Pectin

B. ruminicola 43.3 40.7 1.0 2.6

R. flavefaciens 71.3 29.8 35.5 8.1

Mixed culture 72.6 70.1 52.5 53.0


(From Dehority,


1973)








24

Unidentified cellulose degradation products liberated by

Fibrobacter succinogenes support the growth of the non-

cellulolytic Selenomonas ruminatium (Scheifinger and Wolin,

1973). S. ruminatium decarboxylates the succinate formed by F.

succinogenes to propionate. Decarboxylation of succinate is a

major pathway of propionate formation in vivo.

In recent years this fermentation has been developed into

a process known as anaerobic digestion which was originally

used for treatment of sewage sludges and more recently is

being developed for conversion of energy crops and municipal,

industrial, and agricultural wastes to methane (Richards et

al., 1991). These resources are important both to the future

economy and environmental quality of the world. The methane

produced can be used directly for power generation or upgraded

to pipeline quality levels.

Woody biomass has been previously considered to be highly

refractile to anaerobic digestion without extensive

pretreatment due to the presence of lignin (Zeikus, 1980).

Recent work at the Bioprocess Engineering Research Laboratory

(BERL), Department of Agricultural Engineering at the

University of Florida, has demonstrated that high methane

yields may be obtained from wood without pretreatment, except

particle size reduction (Turick et al., 1991, Chynoweth and

Jerger, 1985, and Jerger et al., 1982). This work has

demonstrated that commercial production of energy through the

biological gasification of woody biomass may therefore








25

represent a viable alternative for energy production in the

United States.

2.11 Cellulolytic Clostridium sp.

Carbon dioxide fixation by photosynthesis yield large

amounts of plant material (Higuchi, 1985) consisting of a

variety of compounds such as cellulose, hemicelluloses, lignin

and other carbohydrates (starch, pectin, chitin) as well as

proteins and lipids. This biomass is decomposed and oxidized

to CO2 and returned to the atmosphere. The microorganisms

degrading this mixture of substances (Rasmussen et al., 1988;

Stoppok et al., 1982; Sleat et al., 1984) encounter a very

complex substrate (Higuchi, 1985). Many microorganisms

interact for mutual benefit, each having specific enzyme

systems for the degradation of certain components of the

complex substrate (Figure 1-1). Clostridium species possess

the enzyme systems necessary to degrade cellulose without

pretreatment (Sleat and Mah, 1989)

In nature, most of the cellulose is degraded to CO2 by

aerobic microorganisms. However, in the global methane cycle

(Atlas and Bartha, 1987), cellulose is also converted to

methane and finally to CO2 in the anaerobic ecosystem.

The cellulolytic organisms which predominate in anaerobic

digesters are anaerobic and sensitive to oxygen; they were not

well known until Hungate (1950) developed roll-tube

techniques.

A cellulolytic anaerobe, Clostridium aldrichii (Pl), has








26

been repeatedly isolated (7X) from a poplar wood-fed

mesophilic anaerobic digester over a period of two years (Yang

et al., 1990). Research indicates the organism is

autochthonous (indigenous) to this system. P1 is the only

cellulolytic bacteria successfully kept in culture from this

digester which is fed primarily poplar wood chips, a potential

energy crop (Jerger et al., 1982). There is a problem in

identification of these anaerobes as they can take 4-8 weeks

to isolate, identify, and enumerate using culture and

microscopy techniques (Madden, 1983).

A recent report (Leschine et al.,1988) described the

isolation and characterization of four anaerobic

nitrogen-fixing cellulose fermenting bacteria from freshwater

mud and soil. Since environments rich in cellulose are

frequently deficient in nitrogen (i.e. peat soils,

agricultural and municipal wastes, and composts)

cellulose-fermenting bacteria that satisfy their nitrogen

requirements through the fixation of Nz would have a strong

selective advantage over those requiring a source of combined

nitrogen. Because vast amounts of cellulose are available in

a wide variety of environments (Hungate, 1950), it is possible

that cellulolytic, nitrogen-fixing bacteria are widespread in

nature and that these bacteria play a significant role in

nitrogen and carbon cycling.

Cellulolytic Clostridium species from decomposing

vegetation (Petitdemange et al.,1984), freshwater sediments








27

(Leschine et al., 1983), estuarine sediments (Madden et al.,

1982), anaerobic digesters (Sleat and Mah, 1985), and human

waste (Wedekind et al., 1988) have been isolated and

characterized. In contrast, very little is known about the

population dynamics of cellulolytic bacteria in these systems.

Studies of the structure-function relationships in these

bacterial cellulolytic systems are revealing new details in

ultrastructural organization of microorganisms (Mayer, 1988).

Gram-positive, spore-forming, cellulolytic rods are probably

more important in anaerobic digesters than in the rumen;

examples are Clostridium lochheadii (Hungate, 1957),

Clostridium lonqisporum (Hungate, 1957), Clostridium

cellobioparum (Chung, 1976), and Clostridium aldrichii (Yang,

1990).

The potential use of these bacteria in the conversion of

lignocellulosic compounds present in municipal and

agricultural wastes into useful products remains to be

explored. The success of monitoring specific populations to

determine which are the important microorganisms in a specific

system and then seeding systems with bacteria to increase

efficiency would potentially save time, money, and resources

in waste treatment.
















CHAPTER 3
THIOTHRIX sp. ANTIBODY PROBE DEVELOPMENT




3.1 Introduction

Research with Thiothrix sp. at the University of Florida

began with the work of Dr. James B. Lackey. His initial work

in this area was to test whether or not Beggiatoa sp. and

Thiothrix sp. were responsive to environmental pollution

changes (Lackey, 1961). His later studies were concerned with

the taxonomy and ecology of the sulfur bacteria particularly

from Florida environmental samples (Lackey et al., 1965). He

repeatedly cited the difficult nature of culturing and

identifying these species. As drip irrigation systems were

installed in central and south Florida citrus groves some

began to cease to function properly because of filter and

emitter clogging. Dr. Harry W. Ford and Donald. P. Tucker at

the Lake Alfred Agricultural Research Center found the most

serious clogging was caused by a sulfur slime formed by

Thiothrix nivea (Ford and Tucker, 1975). While recommendations

have been made to take care of the sulfur slime problem in

drip irrigation systems (Ford, 1979), growth of Thiothrix sp.

and associated pipe blockage continues to be a problem in








29

Florida agricultural systems (Pitts et al., 1990).

Chan-Won Lee in the Department of Environmental

Engineering Sciences, University of Florida (Lee, 1987)

studied Thiothrix sp. with respect to the control of its

overgrowth in the activated sludge process, therefore

inhibiting bulking sludge. After reviewing the literature on

sulfur bacteria it is apparent that Thiothrix sp. are a source

of continuing problems, in particular, environmental systems

as well as in their identification.

The goals of this investigation were: (1) to develop

specific MAB and PAB probes to Thiothrix sp. and (2) use these

probes in immunological procedures with the potential for

Thiothrix sp. detection and identification in environmental

samples.

3.2 Materials and Methods

3.2.1 Microbial Culture Procedures

Thiothrix species types Al, Q, and I, grown on LTH media

(Williams and Unz, 1985), were obtained from Kaye

Shuttlesworth, Department of Civil Engineering, University of

Pennsylvania, University Park. Thiothrix species types 1, 3,

Leucothrix, and type 021N, grown on "I" media (Appendix E)

with Na2S*9H20 at 0.187 g/L substituted for glucose, were

obtained from Michael Richard, Department of Environmental

Health, Colorado State University, Fort Collins. Thiothrix

nivea, grown on C-MY media (Appendix E) made with Warm Mineral

Spring water (Appendix B-2), was contributed by Michael








30

McGlannan, Department of Biological Sciences, Florida

International University, Miami. Beqqiatoa alba, strains B18LD

and B25RD, were sent by John Larkin, Department of

Microbiology, Louisiana State University, Baton Rouge were

also grown on LTH media (Appendix E). Cultures were

transferred every 6-8 weeks. All bacteria were maintained at

room temperature. Transferring these bacteria required

particular care due their "slimey" or "stringy" nature. All

cultures with the exception of Thiothrix type Q and type 021N

were transferred with an inoculating loop. Thiothrix type Q

and 021N required gentle vortexing until filaments were

dispersed. Then 0.5 mls were transferred to 8 mls of fresh

medium. Pure cultures were preserved in 50% sterile glycerol

at -700C.

For assay and immunization procedures pure bacterial

cultures were centrifuged, resuspended in PBS with 1% formalin

and refrigerated. After overnight refrigeration, bacteria were

washed three times with PBS and stored at 40 C.

Quantification of bacterial protein for all bacteria used here

was accomplished by the Lowry method (Lowry et al., 1951).

Bacteria samples used in protein assays were sonicated before

use as previously described (Brigmon, 1987).

3.2.2 Immunization Procedure

BALB/c mice were initially immunized in vivo by

subcutaneous injection with 0.2 ml of Freund's Complete

Adjuvant (Sigma Chemical Co., St. Louis, Missouri) containing








31

100 4l of Thiothrix Al. This was repeated after 2 weeks. At

week 4, the same mice were intraperitoneally injected with 0.2

ml of Freund's Incomplete Adjuvant (Sigma Chemical Co., St.

Louis, Missouri) containing 100 i1 of Thiothrix Al. At week 6,

the mice were injected intravenously with 100 Ul of Thiothrix

Al in PBS. At week 7, a blood sample was obtained from the

tail vein and assayed for antibody production. The two mice

having the highest antibody production were selected for

hybridoma production. At week 12, these mice received 100 pLl

of live Thiothrix Al subcutaneously in 0.2 ml PBS.

3.2.3 Enzyme-linked immunosorbent assay

Formalinized washed Thiothrix sp. or other bacterial

protein were diluted to 10 ig/ml in carbonate-bicarbonate

buffer (Appendix C) and 100 [1l were added to Immulon 2

96-well immunoassay plates (Dynatech, Chantilly, VA.). A

positive control containing only Thiothrix Al and a negative

control containing only E. mirabilis were included on each

plate. Plates were incubated 12 h at 40C. The plates were then

washed 3X with PBST (Appendix C) and then incubated 1 h with

PBSA (Appendix C). All wells were washed with PBST and

duplicate 100 11 of antibody (antisera, hybridoma supernatant,

or ascites preparation) was added to the wells on the assay

plate. Samples of fresh media (HT, Appendix D), wash solution

(PBST, Appendix C), and media from supernatants of cultures of

mouse myeloma cells (SP2/O-AG14) were also employed as

negative controls. The plates were incubated at room








32

temperature for 1 h. After washing 3X to remove unbound

antibody, 100 11 of a 1:1000 dilution in 1% PBSA (Appendix C)

of affinity-purified alkaline phosphatase goat anti-mouse

immunoglobulins (OrgannonTeknika, Malvern, Pennsylvania) were

added to each well. Again the plates were incubated for 1 h

and then washed. One hundred i1 of alkaline phosphatase

substrate (Appendix C), made just before use, were added to

each well. The plates were protected from light and read on a

Titertek Multiskan plate reader (Flow Laboratories, McLean,

Virginia) at 405 nm after 30 minutes. Each plate had controls

of at least two wells without the first antibody, two wells

without the second antibody, and two wells with no antibodies.

Antisera from immunized mice used in the hybridoma protocol

was used as a positive control, and normal mouse serum was

used as negative control. The first column on each plate was

left blank (no antigen or antibodies) to serve as a blank for

the plate reader. Only substrate was added to this column. A

positive result was considered to be an OD of over 0.20 and

over three-fold over background. Five to ten-fold was usually

the case. Any reading below 0.20 was considered negative.

Duplicate samples of hybridoma culture supernatants or

ascites preparations (100 p1) were added to the wells. Serum

from immunized mice, as well as serum from nonimmunized mice

diluted 1:1000 in PBSA were used as positive and negative

controls, respectively. Figure 3-1 is a schematic of the

ELISA.









33



Bacteria are incubated 1 h in ELISA buffer, fixed w/0.25% glutaraldehyde
washed 3X w/PBST then incubated 1 h w/PBSA, then washed 3X w/PBST


Bacteria are incubated 1 h w/ MABs or PABs, then washed 3X with PBST


Bacteria are then incubated 1 h with enzyme-labeled goat anti-mouse
antibody, then washed 3X w/PBST



P1i S


Add substrate, after 30 min incubation plates are read @ 405 nm


Figure 3-1 Schematic of the ELISA protocol.








34

3.2.4 Fluorescent antibody procedure

Pure cultures of Thiothrix types Al, Q, I, and an

activated sludge sample with no Thiothrix sp. present were

fixed for 30 min in PBS with 4% paraformaldehyde in

microcentrifuge tubes (1.5 ml). All bacteria and sludge

samples were washed 3X with PBS. Samples were then incubated

10 min with 1 ml PBS with 10% goat serum. All tubes were

washed 3X with PBS and then incubated 10 min with 1 ml of

select primary antibodies (MABs and PABs) or controls (NMS or

PBS). All samples were again washed 3X and incubated 30 min

with secondary antibody, fluorescein isothiocyanate (FITC)

labeled goat anti-mouse antibody (Sigma, St. Louis, MO.).

After a final wash 3X with PBS samples were examined using a

Nikon epifluorescent microscope. A drop of DABCO (1,4-

diazobicyclo-(2,2,2)-octane (ICN Biochemicals, Costa Mesa,

CA.) in glycerol (Sigma Chemical Co., St. Louis, MO.)) was

added to samples on slides just prior to microscopy to inhibit

photo-fading of the FITC conjugate (Johnson et al., 1982).

Figure 3-2 is a schematic of the FAB protocol.

3.2.5 Generation of Monoclonal Antibodies

Mouse myeloma cells, SP2/0-AG14, were provided by Mrs.

Zelma S. Zam, Department of Ophthalmology, College of

Medicine, University of Florida, Gainesville, Florida. Myeloma

cells were grown in DMEM (Appendix D) supplemented with 10%

fetal calf serum (FCS) and 1.3 10-6 M 8-azaguanine (Sigma

Chemical Co., St. Louis, MO.). After 2 days,the cultures were






















Bacteria are fixed 30 min in PBS W/4% paraformaldehyde, then washed
3X w/PBS and incubated 10 min w/ PBS W/10% Goat Serum


Bacteria are incubated 1 h w/ MABs or PABs, then washed 3X with PBS


Bacteria are then incubated 30 min w/FITC labeled goat-anti mouse
antibody, then washed 3X w/PBS, bacteria were then examined on
an epifluorescent microscope with a drop of DABCO added.


Figure 3-2. Schematic of the FAB protocol.








36

centrifuged at 700 g for 10 minutes and recovered cells were

grown in an incubator in DMEM with 10% FCS. All cell cultures

were grown in an incubator with 5% CO2 air atmosphere at 370C

with 90% relative humidity. Colcemid (Sigma Chemical Co., St.

Louis, Mo.), was added to the culture media (10 pg/ml) 48

hours prior to the hybridization procedure to arrests cells in

the M phase of cell division which enhances hybridoma

production (Miyahara et al., 1984). Myeloma cells and the

splenocytes from the hyperimmunized mice were fused at a 1:10

ratio by a variation of the method of Kohler and Milstein

(1975). One ml of 50% polyethylene glycol (PEG) (Sigma

Chemical Co., St. Louis, Mo., molecular weight 1450) with 15%

dimethyl sulfoxide (DMSO) (Fisher Scientific, Fairlawn, New

Jersey), pH 8.0 at 400C was added over a 1 min interval to

pelleted myeloma cells and splenocytes. The cell-PEG mixture

was incubated for 1 min at 400C. DMEM (Appendix D) was added

to the suspension over 3 min at the rate of 10 ml/min.

The cell suspension was centrifuged at 700 g for 10 min

and resuspended in 35 ml of HAT (Appendix D). The suspension

was then distributed (100 1l/well) into 4-96 well culture

plates (NUNC, Denmark). The cells were incubated as described

previously and fed with 50 Il of HAT every 5 days. After 2

weeks in the HAT medium, 100 ~1 of supernatant from actively

growing hybridoma cell cultures was removed. At this time 100

1l of DMEM supplemented with 10% FCS containing HT

(Appendix D) was added to all wells in the culture plates.








37

Thereafter the cultures in the wells were fed every 5 days

with 50 i1 of the HT mixture for ten days.

3.2.6 Specificity Assay

Once monoclonal antibodies were established their

specificity was determined. The ELISA procedure was performed

testing a panel of 14 bacteria including 6 Thiothrix sp. All

bacteria were tested at the same protein concentration in

ELISA buffer (10 [g/ml) in duplicate on the ELISA plates.

3.2.7 Determination of Isotype of Monoclonal Antibodies

In order to determine isotype or immunoglobin type of the

MAB the ELISA procedure was performed using class- (IgG, IgA,

IgM) and subclass-specific (IgG1, IgG2a, IgGb, IgG3) affinity

purified alkaline phosphatase labeled goat anti-mouse

immunoglobulins (Organnon Teknika, Malvern, Pennsylvania).

MAB isotype controls employed were MABs specific for

Actinobacillus actinomycetemcomitans and Hemophilus

aphrophilus supplied by Dr. William McArthur, Department of

Oral Biology, College of Dentistry, University of Florida.

3.2.8 Quantitative Absorption

Formalinized washed Thiothrix sp. or other bacterial

protein at different concentrations (10, 1.0, 0.01, and 0.001

and 0 jIg/ml) were pelleted in microfuge tubes. One ml of

monoclonal antibodies diluted to 1:500 in PBSA were added to

the appropriate tubes. A control with no bacteria present was

set up for each MAB. The pellet was tapped to resuspend it and

the mixture was incubated at 410C overnight. The next morning








38

the suspension was microfuged and the supernatant was tested

by ELISA. The antigen on the plate was known to react with the

antibody being tested (Thiothrix Al). One of the absorbing

bacteria used was one known not to cross react with the

antibodies Leucothrix).

3.2.9 Sensitivity Assay

In order to test the lower threshold of assay sensitivity,

pure cultures of the microorganisms were grown and tested with

Thiothrix type Al protein at concentrations ranging between

(0.00-144.3 [lg/ml) in the ELISA procedure.

3.2.10 Testing for Carbohydrate Epitopes

Periodate oxidation was used in conjunction with the ELISA

procedure in order to determine if MABs are specific for

carbohydrate or non-carbohydrate determinants (Woodward et

al., 1985). Briefly, two plates were prepared for the ELISA

with Thiothrix Al as previously described after incubation

with PBSA. Plates were washed with 50 mM sodium acetate buffer

(pH 4.5). One washed plates were then exposed to 200 Ll/well

10 mM sodium meta-periodate (Sigma Co., St. Louis, MO) in 50

mM sodium acetate buffer, pH 4.5, for 1 h at 250C in the dark.

Following a brief rinse with 50 mM sodium acetate both plates

were then incubated with 200 Ll/well 50 mM sodium borohydride

(Fisher Scientific, Fairlawn, NJ) in PBS for 30 min at 250C.

Both the periodate and borohydride were prepared just prior to

use. Following 3 washes with PBST the ELISA procedure

continued with both plates (periodate and non-periodate








39

treated) as previously described with the addition of MAB

preparations.

3.3 Results

3.3.1 Probe Development and Specificity

Supernatants from cell cultures showing significant growth

after 14-21 days were tested for antibody production to

Thiothrix Al by ELISA. Those cultures showing positive

antibody activity were expanded and later retested. Those

cultures showing consistent results were cloned by limiting

dilution, reidentified, cloned a second time and retested by

ELISA using the immunizing bacteria at the same concentration.

The positive monoclonal antibodies were further tested against

other Thiothrix species and other bacteria at a similar

concentration.

Table 3-1 presents data of selected species specific

monoclonal and polyclonal antibodies tested with various

bacteria. Positive results are OD's > 0.20. One of the MABs,

16010, showed some reactivity with other bacteria besides

Thiothrix sp.. The other three preparations, the MABs 3511 and

9168 as well as the PAB, were specific for Thiothrix sp.

particularly the immunogen Thiothrix Al.

3.3.2 Purification of Monoclonal Antibodies

Once hybridoma cells producing specific antibody for

Thiothrix sp. were established, cells were passage in BALB/c

mice. Briefly, pristane primed mice were injected with 106

hybridoma cells producing specific antibody and the












Table 3-1. ELISA screening of specific MABs for specificity of
binding to select bacteria. MABs and PABs were
tested for cross reactions with other Thiothrix
species, select filamentous bacteria, and various
bacteria found in environmental samples.



ABSORBANCE (405 nm)*

Hybridoma Number

Microorganism 16010 3511 9168 PAB



Thiothrix Al 2.32 2.35 2.50 2.38
Thiothrix I 0.61 0.63 2.36 0.44
Thiothrix nivea 0.90 0.73 0.62 0.50
Thiothrix Q 0.56 0.62 2.42 0.47
Thiothrix TH1 0.89 1.17 1.75 0.77
Thiothrix TH3 1.10 0.54 1.52 0.73
Type 021N 0.55 0.15 0.03 0.17
Beqgiatoa alba 0.40 0.10 0.01 0.12
(B18LD)
Beqiatoa alba 0.22 0.08 0.02 0.09
(B25RD)
Leucothrix sp. 0.50 0.10 0.08 0.18
Desulfovibrio 0.55 0.00 0.01 0.15
desulfuricans
(ATCC 29577)
Enterobacter 0.60 0.10 0.20 0.19
aerogenes
(ATCC 13048)
Escherichia coli 0.10 0.03 0.01 0.18
Pseudomonas 0.15 0.15 0.03 0.18
aeroginosa


*Absorbance > 0.20 considered positive value








41

ascites fluid collected. The antibody was precipitated with

45% saturated ammonium sulfate and dialyzed against multiple

changes of PBS over 48 h. Ascites fluids were frozen with

antiserum samples at -70C. A concentration of 1:1000 was used

for assays with MABs and PABs.

3.3.3 IsotypinQ Monoclonal Antibodies.

Clones 9168 and 3511 were found to be of the IgG2a type

and 16010 was an IgM type immunoglobin as summarized in Table

3-2. All three were comprised of kappa chains. There was no

cross-reactivity with the isotype controls.

3.3.4 Quantitative Absorption Assay.

A quantitative absorption was preformed to verify the

specificity of the monoclonal antibodies. Thiothrix Al and

Leucothrix sp. were used as absorbing bacteria at various

concentrations. The antibody activity in the selected

hybridoma ascites preparations was removed in a dose-dependent

manner by absorption with Thiothrix Al, but not removed after

absorption with Leucothrix sp. Most antibody activity was

absorbed out completely by 10 [g/ml of Thiothrix Al, as

indicated by an OD of less than 0.20 (Table 3-3).

3.3.5 Specificity for Carbohydrate Epitopes

The antigenic reactivity of MABs 16010 and 3511 was

destroyed when Thiothrix Al coated ELISA plates were subjected

to sodium meta-periodate oxidation (Table 3-4). However MAB

9168 demonstrated decreased yet still positive activity (Table

3-4). Plates tested simultaneously without periodate were











Table 3-2. ELISA isotype testing of monoclonal antibodies
developed against Thiothrix sp.


Positive
Hybridoma Immunizing Binding Data
Number Isotype Antigen (ELISA, OD 405 nm)


9168 IgG2a Thiothrix Al Thiothrix Al
Thiothrix P*

3511 IgG2a Thiothrix Al Thiothrix Al
Thiothrix P*

16010 IgGM Thiothrix Al Thiothrix Al
Leucothrix sp.
Thiothrix P*

AA1 IgGi A. actinomycetemcomitans None

AA2 IgG2a A. actinomycetemcomitans None

AA3 IgG3 A. actinomycetemcomitans None

HA IgM H. aphrophilus None

* Fresh environmental Thiothrix sp. from City of Palatka.













Table 3-3.


Quantitative absorption of specific monoclonal
antibodies with ELISA*. A constant dilution of MAB
3511 and MAB 9168 was incubated with various
concentrations of Thiothrix type "Al" and
Leucothrix sp. and then assayed with Thiothrix type
"Al" treated plates


ABSORBANCE @ 405 nm**

Bacterial Thiothrix Leucothrix Thiothrix Leucothrix
Concentration + + + +
(Li/ml) 9168 9168 3511 3511

10.000 0.01 2.60 0.02 2.40
1.000 0.78 2.46 0.60 1.86
0.100 1.24 2.60 1.66 1.59
0.010 2.29 2.30 1.65 1.88
0.001 2.09 2.20 1.81 2.58


*Absorbances of controls with MAB 9168 and MAB 3511 were all
> 2.00.
**Average of 2 readings from each of 2 separate determinations.















Table 3-4. Effect of periodate oxidation of Thiothrix Al on
the binding of MABs 16010, 3511, and 9168



Treatment MAB OD405a


Controlb 16010 1.64
10 mM 16010 0.03
Control 3511 1.85
10 mM 3511 0.08
Control 9168 2.20
10 mM 9168 0.20



a Average of 2 readings from each of 2 separate
determinations.
b No periodate oxidation but treated with buffer (acetic acid-
sodium acetate at pH 4.5) followed by sodium borohydride
reduction.








45

controls. These results indicate that the MABs developed here

recognize carbohydrate-containing epitopes associated with

glycoproteins or glycolipids or both on the surface of these

bacteria. MAB 9168 will be looked at further. Positive samples

are OD's greater than 0.20.

3.3.6 Fluorescent Microscopy.

Fluorescent photomicrographs of Thiothrix type "Al"

(Figure 3-3) and type "Q" (Figure 3-4) using 3511 at a 1:50

dilution are shown. Not shown are controls which are

essentially dark photographs.

3.3.7 Light and Phase Microscopy

Leucothrix type N2 is shown in Figure 3-5 using brightfield

microscopy at 400X. Leucothrix sp. are filamentous bacteria

which form rosettes although they do not deposit sulfur

intracellularly environments (Brock and Madigan, 1991).

Type "021N" is shown in Figure 3-6 using the modified gram

stain (Appendix A) at 400X. It's unusual physical

characteristics which are evident here have been reviewed by

Williams et al. (1987). These characteristics include clusters

of large swollen cells or bulbs in cultures with a nonlinear

arrangement of cells, with crosswalls at odd angles to the

longitudinal axis of the filament. The morphology of the

bacteria seems to change with the age of the culture. Ultra

structural studies have revealed unusual double-layered

membrane internal to the cytoplasmic membrane present on only

one side of the cell (Williams et al., 1987).




























































Figure 3-3 Fluorescent inmunoassay rsting rhiothrix type Ai
with MAB 3511 at 400C.


























































































-- a -,:: -,


S- :'^-s2 s,%- il uncsS -s
- M.-E-


- 1, j -























































Figure 3-5. Leucothrix type N2 with bright field microscopy at
400X.


















































.4)

























t -I
---


ifLd -ram stain


~








50

Thiothrix type "3" treated with the "sheath stain"

(Appendix A) are seen in Figure 3-7 at 400X. As described by

Jenkins et al. (1984) the sheaths are clear to pink and cells

stain purple.

Thiothrix type "Q" with modified gram stain at 400X

reveals the filamentous nature of this species (Figure 3-8).

This species is hard to transfer without the entire culture

coming with it due to its "stringy" or cohesive nature as

previously reported by Williams and Unz (1985).

3.3.8 Sensitivity assays

Based on their high specificity, MABs 9168, 3511, and the

PAB preparation were tested for sensitivity in the ELISA to

the immunogen Thiothrix type Al. The results of these tests

are shown for these probes in Figures 3-9, 3-10, and 3-11

respectively. Results are from duplicate samples in the ELISA

as previously described. Logarithmic transformations were used

to fit the curves. All three MAB preparations showed high

correlation (P < 0.01) with respect to absorbance in the ELISA

as a function of Thiothrix Al protein applied to the ELISA

plates.

3.4 Discussion

Sensitive and specific MABs and PABs directed against

Thiothrix type "Al" were developed in the laboratory.

Initially Thiothrix type Al was selected as the immunizing

agent because no other filamentous culture was available for

testing. P. mirabilis was initially used as a negative









































r e
I ~7 ?':


































Figure ;-8. Thic-hrix type Q treated with modified Gram stain
400 ::.


- 13
























R^2 = 0.966


2 3 4 5


Thiothrix Al


protein log ng/ml


Figure 3-9. Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with clone
9168
























R 2 = 0.937


2 3 4 5


Thiothrix


Al protein


log ng/ml


Figure 3-10. Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with
clone 3511

























R^2 = 0.923


2 3 4 5


Thiothrix Al


protein log ng/ml


Figure 3-11. Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with
PAB








56

control. The three hybridomas which were found to be suitable

following the initial screening were used for testing in

addition to PABs obtained from mice used in the fusion

procedures.

Antibody preparations were tested in a panel of 14

bacteria including 6 Thiothrix sp., 4 different filamentous

bacteria, and 4 other bacteria which could be in environmental

samples. One of the monoclonal antibodies, 16010, showed some

reactivity with several other bacteria besides Thiothrix sp.

and therefore was not used in further testing. MABs 9168 and

3511 were found to be of the IgG2a type while MAB 16010 is an

IgM type immunoglobulin. All three were found to be specific

for carbohydrate antigenic determinants on Thiothrix type Al.

Highly specific and sensitive monoclonal and polyclonal

antibodies were developed and calibrated with the ELISA. The

FAB procedure employed was a detection procedure with direct

observation of fluorescent-antibody labeled filaments. The

MABs were highly specific such that only Thiothrix sp. were

positive in the ELISA with bacteria tested here (Table 3.1).

As little as 0.56 [g/ml Thiothrix Al protein could be detected

using the ELISA and the probes 3511, 9168, and PAB. Bacterial

protein was the best means of quantifying ELISA sensitivity

due to the complex physical characteristics of the filamentous

Thiothrix sp.. These characteristics include variable filament

length and the cohesive nature of the cultures. The sonicated

samples were easier to pipette evenly in the assay plates than








57

whole Thiothrix sp. bacterial cultures.

The sensitivity of the ELISA described here is comparable

to those described for Salmonella detection (Lee et al., 1990)

using protein from whole bacteria as an indices of

sensitivity. Much higher sensitivity is possible using ELISA

procedures with purified antigens in the range of 100 pg to 1

ng/ml (Herrman, 1986). It has been reported that using an

enhanced developing system that amplifies the signal to noise

ratio of the quantitatively bound second antibody can enhance

an ELISA's sensitivity such that as little as 1 to 10 pg of a

specific protein can be detected (Macy et al., 1988).

For the purpose described here the cross-reactivity and

sensitivity are sufficient for detection in environmental

samples. No other known immunological work has been published

with Thiothrix sp. at the time of this writing. Future work in

this area would be identification of the specific antigen or

epitope with which the MABs are reacting. It would be possible

then to extract the antigen (e.g. lipopolysaccharide) and

increase the sensitivity of the ELISA. In addition, use of the

amplification techniques mentioned above would enhance the

assay sensitivity. As more pure cultures of Thiothrix sp.

become available it would be useful to compare reactivity with

these probes and employ them as part of a typing system.










CHAPTER 4
Thiothrix sp. DETECTION IN ENVIRONMENTAL SYSTEMS


4.1 Introduction

Thiothrix sp. have a worldwide distribution in selected

environments from sulfide rich springs in Russia (Winogradsky,

1888) and the United States (Lackey et al., 1965) to sewage

treatment plants in Japan, South Africa, and Europe (Jenkins

et al.,1984). Thiothrix sp. live in a gradient in the

environment, existing in flowing water in which the sulfide

gradient is about 0.1 to 1.0 mg/liter, the oxygen

concentration is about 10% or less of saturation, and the pH

is near neutrality (Larkin and Nelson, 1987). They have also

been found attached to mayfly larva in sulfur springs (Larkin

et al., 1990). Leucothrix sp. have been found attached to

marine animals (Brock, 1991).

The presence of Thiothrix sp. in nature is typically

associated with its attachment to leaves, rocks, or algae by

the direct observation of the holdfast, sheath, cellular

elements, and intracellular sulfur globules which are

characteristic of this genus (Caldwell et al., 1975). Under

favorable conditions, Thiothrix sp. may dominate a particular

niche appearing as a near monoculture in nature (McGlannon and

Makemson, 1990), thus simplifying identification.

In drip irrigation systems (Ford and Tucker, 1975) and

citrus drainage systems (Spencer et al., 1963) growth of








59

filamentous Thiothrix sp. can cause physical blockage of

water. This blockage in pipes and valves is typically in

association with iron precipitating bacteria (Ford and Tucker,

1974) which can form a "slime". Prevention and detection of

Thiothrix sp. problems is difficult in these systems until

blockage occurs and treatment depends on water analysis and

monitoring (Pitts et al., 1990). Once pipes, valves, or

emitters are blocked they have to be physically replaced. This

can be costly in terms of repair as well as potential crop

damage and wasting resources.

Thiothrix sp. are also frequently associated with

activated sludge bulking (Farquhar and Boyle, 1971a & 1971b).

While methods to identify most of the problem filamentous

microorganisms exist, they require a certain degree of skill

and experience (Jenkins, et al., 1984). Thus there is a need

for methods of rapid identification of causative

microorganisms and documenting the effectiveness of methods

used for their control. The collection of such information

should permit the diagnosis and solution of most bulking

problems in activated sludge.

4.2 Materials and Methods

The primary source of activated sludge samples was a

contact stabilization process treating domestic and hospital

wastewaters from the University of Florida campus (UF plant).

Additional samples were obtained from a completely mixed

process treating domestic and commercial wastewaters from the








60

City of Gainesville, Florida, Main Street and Kanapaha plants.

Mr. Bud Roselle, an operator at the Main Street Plant was

helpful in sampling and volunteering information. Mr. David

Welch, a supervisor at the Kanapaha Plant, was supportive of

this work.

Samples of blocked irrigation systems with Thiothrix sp.

were obtained from the IFAS extension office, Plant City,

Florida. Scanning electron micrographs of pipe samples are

courtesy of Dr. Henry Aldrich, Department of Microbiology,

University of Florida.

Warm Mineral Spring samples were obtained in November,

1990. Archaeologist Wilburn "Sonny" Cockrell, director of

Florida State University's Warm Mineral Springs Archaeological

project was helpful in sampling and information. Samples from

Orange Springs cave and spring run were obtained from Mr. Tom

Morris, Gainesville, Florida, a professional scuba-diver. Mr.

Morris is also responsible for the underwater photographs.

Thiothrix sp. samples from the City of Palatka water storage

tanks were obtained in October, 1991.

Water samples were obtained from Orange Springs, Warm

Mineral Springs, and the City of Palatka at the same time

Thiothrix samples were collected. These water samples

(chemical analyses are shown in Appendix B), were used in

culture, enrichment and storage procedures for Thiothrix sp.

The spring water was used in place of distilled water in LTH

medium preparation. In addition, autoclaved spring water was








61

added to samples for storage and microscopy work.

4.2.1 Thiothrix detection in mixed cultures with ELISA

Due to the complex nature of environmental samples, it

would be unlikely to obtain a pure Thiothrix sp. culture after

enrichment with selective media. The ability of the assay to

detect Thiothrix Al, added to mixtures of 10% sludge (w/v)

which were negative for Thiothrix sp. (from the City of

Gainesville, Kanapaha Plant) was tested with the ELISA.

4.2.2 Environmental Sampling Procedures

Samples collected from all sites were placed on ice

immediately and processed in the laboratory within 24 h. In

most cases on local sampling trips the time was 1-2 h. Smaller

samples were collected in sterile 50 ml polypropylene tubes

(Costar Corp., Cambridge, MA). Water samples were collected in

sterile 1 L polycarbonate containers (Corning Inc., Corning,

NY). Samples were collected in water from the specific site

and kept in that water until processed in the laboratory.

4.2.3 Identification of Thiothrix in environmental samples

Samples thought to contain Thiothrix sp. were identified

according to the methods described by Jenkins et al., 1984.

Briefly, this involved initial observation with phase-contrast

microscopy, followed by the "S" test to check for sulfide

uptake (Jenkins et al., 1984) which would be indicated by the

presence of sulfur-containing granules in the filaments.

"Twitching" or waving motility characteristic of this species

would also factor in identification. Confirmation of samples








62

was accomplished by the modified Gram and sheath stains

(Appendix A). Samples were also enriched for 24-72 h using

either LTH media made with water from Palatka, Warm Mineral

Springs, or Orange Springs (See Appendix B), or just one of

the water samples (autoclaved) and then observed with darkfiel

microscopy. Samples which were stored or saved long term

(1 year) were kept in Warm Mineral Springs water.

4.2.4 Fluorescent antibody procedure

Activated sludge, spring samples, Thiothrix Al, and

activated sludge with no previously observed Thiothrix sp.

present were fixed for 30 min in PBS with 4% paraformaldehyde

in microcentrifuge tubes (1.5 ml) or heat fixed on microscope

slides and incubated for 10 min in paraformaldehyde. All

preparations were then washed 3X with PBS. Samples were then

incubated 10 min with 1 ml PBS with 10% goat serum. All tubes

and slides were again washed 3X with PBS and then incubated 10

min with 1 ml of select antibodies (MABs and PABs) or controls

(PBST or PBSA) diluted 1:50 in PBS. Samples were again washed

3X and incubated 30 min with secondary antibody, FITC labeled

goat anti-mouse antibody (Sigma, St. Louis, MO.) diluted 1:50

in PBS. After a final wash with PBS (3X) samples were examined

using a Nikon epifluorescent microscope. Preparations made in

microcentrifuge tubes stayed fluorescent for 5-7 days stored

in the dark at 40 C. DABCO was added to samples prior to

examination (Johnson et al., 1982).








63

4.2.5 ELISA of Environmental Samples

Samples which were observed to contain Thiothrix sp.

samples were stored in Warm Mineral Spring Water at 4C until

assayed. This storage kept the cultures viable and healthy for

up to 1 year. Prior to the assay, samples were washed 3X with

ELISA buffer and then adjusted to pH 9.8 to the original

volume. Samples were then pipetted onto ELISA plates in

duplicate as previously described. Controls included

environmental samples with no primary and samples with no

secondary antibody on each plate. Positive controls with

Thiothrix Al were on every plate.

4.3 Results

4.3.1 Thiothrix detection in mixed culture with ELISA

The three calibrated probes tested showed significant

correlation of PAB and MAB reactivity in the ELISA with

Thiothrix Al protein mixed with 10% (w/v) with Thiothrix sp.

negative activated sludge (Figures 4-1, 4-2, 4-3). PAB showed

a higher correlation (Figure 4-1) than in testing pure culture

(3-9). Both MABs 3511 (Figure 4-2) and 9168 (Figure 4-3) ELISA

results did show lower correlations although still significant

(P < 0.05) and higher background in mixed culture as compared

to pure culture (Figures 3-7 and 3-8).

4.3.2 Identification of Thiothrix in environmental samples

No Thiothrix sp. were detected from any samples obtained

monthly over a 12 month period from the City of Gainesville

Kanapaha treatment plant using ELISA, FAB, or microscopy
























R^2 = 0.984


2 3 4 5


Thiothrix Al


protein log ng/ml


in 10% sludge


Figure 4-1. Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with PAB
in 10 % sludge (w/v)


(w/v)

























R^2 = 0.884


2 3 4 5


Thiothrix Al protein log ng/ml in 10 % sludge (w/v)












Figure 4-2. Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with MAB
3511 in 10 % sludge (w/v)






















R^2 = 0.879


2 3 4 5 6

Thiothrix Al protein log ng/ml 10% sludge (w/v)













Figure 4-3. Relationship between absorbance and concentration
of Thiothrix Al protein using the ELISA with MAB
9168 in 10 % sludge (w/v)








67

methods (Jenkins et al., 1984). Thiothrix sp. were detected in

9 of 12 samples from the City of Gainesville Main Street

treatment. There was a new section being built at the time

but all samples were from the "East" or "Old" side as the

operators referred to it. Thiothrix samples were found in 6 of

12 samples over the same 12 month period at the UF plant.

None of the plants were ever sampled under bulking

conditions for this study. On November 19, 1991 upon arriving

to the Main Street Plant, I was informed that a load of oil

had been apparently dumped into the system causing the system

to be monitored constantly to prevent disruption of operation.

Large quantities of Thiothrix sp. were observed in the system

that sampling day.

Figure 4-4 shows a Thiothrix sp. from the UF plant at 400X

using darkfield microscopy after the "S" test has been

applied. Enriching samples in LTH medium made with Warm

Mineral Spring Water enhanced detection of Thiothrix sp. as

shown in a sample from the Main Street Plant (Figure 4-5).

This darkfield micrograph at 100X shows cross-bridging

occurring between floc particles. Figure 4-6 shows the classic

limited "twitching" or swaying motion at 100X used to help

identify Thiothrix sp. in an enriched sample from the Main

Street Plant. This motility helps differentiate Thiothrix sp.

from two other filamentous bacteria which also deposit sulfur

intracellularly, type 021N and Beqqiatoa species.



























































Figure 4-4. Thiothrix sp. in sulfide enriched activated sludge
sample from the UF plant 400X






































AA)k
a:


Figure 4-5. Thiothrix sp. in LTH with WMS water enriched
activated sludge sample from the Main Street plant
400X showing signs of "cross-bridging floc"
particles



























































Figure 4-6. Thiothrix sp. in LTH -.ith V\MS water enriched
activated sludge sample from the Main Street plant
400X showing signs of "twitching" or swaying
motility









71

A classic rosette in a fresh sample of Thiothrix nivea

from Warm Mineral Spring at 1000X with brightfield microscopy

is shown in Figure 4-7. Figure 4-8 shows an older sample of

Thiothrix nivea with the sheath stain at 400X. Figure 4-9

compares a Thiothrix nivea filament next to much larger

Beqqiatoa gigantea filaments from Warm Mineral Springs at 100X

(identification of Beggiatoa gigantea with the assistance of

Dr. John Larkin, Louisiana State University, Baton Rouge).

Thiothrix nivea seemed to dominate near the head of the spring

where the smell of H2S was the strongest, while Begqiatoa

gigantea was most prolific 50 yards further down the spring

run.

In October, 1991, Mr. William Knotts, an operator at the

City of Palatka, Florida water storage facility, noticed a

"hollow" sound while walking across the top of a 500,000

gallon water storage tank. On closer examination of the inside

of the tank it was evident that there was serious corrosion in

the roof of the tank. Dr. Henry Aldrich of the Department of

Microbiology and Cell Science, University of Florida, was

called in as a consultant by Mr. Ted Crom, Crom Corporation.

Figure 4-10 shows the aeration system on top of a water

storage tank at the City of Palatka water storage facility.

All the white material was a profuse Thiothrix nivea growth.

Some of the filamentous growths were 6 feet long. Figures 4-11

and 4-12 are darkfield photomicrograph at 100X with different

lighting of the bacteria from the aerator. It appeared to be































































































figure -7. Thicthrix niv'ea rosette :rcm ',arm:iu-~erai S~rings
at 1000X.


.
cu




















\ SR


Figure =-3. Thiothrix nivea frc- -:arm Mneral Sorinas
at 4OOX with sheath .tain.


NA

























































Figure 4-9. Thiothrix nivea next to filaments of Beaqiatoa
aqiantea from Warm Mineral Springs ilOOX)





























































Figure -i0. Thiothrix nivea rrow;th or "bloon" in City of
Palatka Twater storage tank aeration system


























































Figure 4-11.Thiothrix nivea darkfield micrograph (100X) of
fresh sample from City of Paiatka water storage
tank aeration system















Ar


!4
,,4


Figure 4-12. Thiothrix nivea darkfield micrograpn 100X) of
fresh sample from City of Palatka .-atr storage
tank aeration system with different -ighting


i ~c,








78

a pure culture. Beneath the white Thiothrix covered growth

there did appear to be some algae which the Thiothrix nivea

was attached to. Each large Thiothrix nivea "strand" seemed to

have an algae "core". Figure 4-13 is a darkfield micrograph

at 400X of Thiothrix nivea attached to algae. Thiothrix sp.

samples were also obtained from Orange Springs by Mr. Tom

Morris who was doing some professional diving in this private

spring. I received samples from 60 feet underground as well as

from the spring mouth and run. A photograph Mr. Morris took in

the cave of some Thiothrix sp. growing by some "sulfur seeps"

as they are refereed to are evident in Figure 4-14. A phase

contrast photomicrograph (Figure 4-15) shows Thiothrix sp.

growing with algae in a sample from the spring run. Showing

similar characteristics in samples from Warm Mineral Springs,

Figure 4-16 shows Thiothrix sp. attached to diatoms in a

phase-contrast photomicrograph.

Pipe samples sent by a farmer to a county extension agent

from Polk county Florida were brought to the Department of

Microbiology and Cell Science. On observation by scanning

electron microscopy (SEM) it was revealed that the pipes were

blocked by a thick growth of Thiothrix sp. Apparent blockage

of the irrigation system by Thiothrix sp. caused crop damage.

Half of the green pepper plants died in a field as a result of

a metered amount of water being pumped into the system but

only a portion of the plants were getting the water.



























































Figure --13. Thiothrix nivea attached to algae in darkfield
micrograph (400X) of fresh sample from City of
Palatka water storage tank



















-r. v^'.^
It91 -^" T
,^ r c-" ''r r *


Figure 4-14. Thiothrix sp. at sulfur seeps in Florida Orange
Spring at a depth of approximately 40 feet. Photo
by Mr. Tom Morris.























\t


*0
p


I


t.


Figure 4-15. Thiothrix sp. in spring run at Florida Orange
Spring attached to Charms (Phase Contrast, 400X)


:6


p r-.


































4 .4





f*i
. ..^ -^________


Figure 4-16 Thiothrix sp. attached to diatoms at Warm Mineral
Springs (Phase Contrast, 400X)








83

Figure 4-17 is a scanning electron micrograph (SEM) of a

section of the blockage (SEM by Dr. H. Aldrich, Department

Microbiology and Cell Science).

4.3.3 Fluorescent Microscopy of Environmental Samples.

A phase-contrast photomicrograph of FAB-treated sample

from the Main Street Plant on November 19, 1991 is shown in

Figure 4-18. These samples were enriched 3 days in the LTH-

Warm Mineral Spring water prior to the FAB procedure. This was

a day there had been an apparent oil spill into the wastewater

and the plant was having problems. Figure 4-19 shows the same

sample only with fluorescence. This was the only day

Thiothrix rosettes were observed in a sewage treatment plant

in the course of this study. Figure 4-20 shows another sample

from the same day using the FAB procedure.

Figure 4-21 is a phase-contrast photomicrograph from a

sample taken from Orange Spring treated with the FAB assay.

This samples were assayed the day they were received, which

was the day after sampling. Figure 4-22 is the same field of

view only with fluorescence. Figure 4-23 is an FAB treated

sample from Warm Mineral Springs. This sample was enriched in

the LTH-Warm Mineral Spring procedure. In both Figures 4-22

and 4-23 a large amount of background fluorescence is evident

from naturally occurring pigments at the two separate sites.

Autofluorescence in these samples came from pigments including

cytochromes in plant, algae, and certain eucaryotes.





























































Figure -17. Thiothrix sp. n pie ...hich caused irrigatin
system blockage and subsequent crop damage in
Polk County, Florida. SEM by Dr. Henry Aldrich,
Department of :-icccbio-ocv and Cell Science



























































Figure 4-18. Main street plant sample of activated sludge with
numerous filamentcus bacteria seen at 100X on
light microscopy.



























































Figure 4-19. Main street plant sample of activated sludge with
numerous filamentous bacteria seen at 100X with
fluorescent microscopy having been treated with
the fluorescent immunoassay using MAB 3511




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