EPA-600/2-75-032
September 1975
Environmental Protection Technology Sen,
BIOFLOCCULATION AND THE
ACCUMULATION OF CHEMICALS BY
FLOC-FORMING ORGANISMS
Municipal Environmental Research Laboratory
Office o! Research and Development
U.S. Environmental Protection Agency
Cincinnati. Ohio 45268
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
U. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY STUDIES series. This series describes research
performed to develop and demonstrate instrumentation, equipment
and methodology to repair or prevent environmental degradation from
point and non-point sources of pollution. This work provides the
new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22151.
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EPA-600/2-75-032
September 1975
BIOFLOCCULATION AND THE ACCUMULATION OF
CHEMICALS BY FLOC-FORMING ORGANISMS
Patrick R. Dugan
Department of Microbiology
Ohio State University
Columbus, Ohio U3210
Grant No. 17050 DFJ
Program Element No. 1BBOU3
Project Officer
Cecil W. Chambers
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 1+5268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 1*5?68
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products con-
stitute endorsement or recommendation for use.
ii
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FOREWORD
Man and his environment must be protected from the adverse effects
of pesticides, radiation, noise and other forms of pollution, and
the unwise management of solid waste. Efforts to protect the
environment require a focus that recognizes the interplay between
the components of our physical environment—air, water, and land.
The Municipal Environmental Research Laboratory contributes to this
multidisciplinary focus through programs engaged in
t studies on the effects of environmental contaminants on
the biosphere, and
0 a search for ways to prevent contamination and to recycle
valuable resources.
Biological floe formation is an essential activity in the biological
conversion of soluble organic waste material to insoluble settleable
solids. A proper understanding of bioflocculation may lead to
improvement in the activated sludge process. This report discusses
the relationship of bioflocculation to waste treatment with special
emphasis on the adsorption capacity of biopolymers for soluble and
insoluble mineral and organic particles.
A. W. Breidenbach, Ph.D.
Director
Municipal Environmental
Research Laboratory
iii
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ABSTRACT
Several floe-forming bacteria were isolated from polluted water
by this and other laboratories. All organisms studied produced
extracellular polymer fibrils that were related to flocculation.
The extracellular polymers have high adsorption capacity for:
soluble metal and other mineral ions, soluble organic nutrients
(BOD), soluble toxic organics, insoluble mineral particles and
insoluble organic particulates. The bacteria remove BOD by
physical adsorption as well as by oxidative metabolism and can
convert oxygen demanding organics to more extracellular polymer.
Production of polymer can be stimulated nutritionally to yield
amounts that have waste treatment-pollution abatement potential
on a commercial scale. The relationship of bioflocculation to
waste treatment and lake eutrophication is discussed and the
basic mechanism of bioflocculation is considered. Biochemical
activity of individual floe-forming cells is examined because
of its relevance to polymer synthesis. Taxonomy of floe-formers
is also considered in relationship to biochemical activities.
This report was submitted in fulfillment of Grant Number 17050 DFJ,
by the Ohio State University, under the partial sponsorship of the
Office of Research and Development, Municipal Environmental Research
Laboratory, Environmental Protection Agency. Work was completed as
of June 19T1.
IV
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CONTENTS
Sections Page
DISCLAIMER ii
FOREWORD iii
ABSTRACT iv
LIST OF TABLES vi
LIST OF FIGURES viii
ACKNOWLEDGMENTS xii
I CONCLUSIONS 1
II RECOMMENDATIONS U
III INTRODUCTION 5
IV MATERIALS AND METHODS 12
V EXPERIMENTAL RESULTS AND DISCUSSION 29
VI GENERAL DISCUSSION OF THE IMPLICATIONS OF MICROBIAL
POLYMER SYNTHESIS IN WASTE TREATMENT AND LAKE
EUTROPHICATION 116
VII REFERENCES 126
VIII LIST OF PUBLICATIONS 13U
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TABLES
No.
Page
1 Floe-forming isolates, source of isolation and
reference to information if available 30
2 Biochemical reactions of floe-forming bacteria 31
3 Reaction of floe-forming bacteria in sugar broth with
bromothymol blue indicator 32
k Reaction of floe-forming bacteria in alcohols and sugar
alcohols using bromothymol blue indicator 33
5 Biochemical characteristics of Zoogloea isolate No. 1
and Zoogloea ramigera isolate No. 115 3^
6 Growth of isolates No. 1 and No. 115 in basal medium
II plus amino acids as carbon and nitrogen sources 35
7 Growth of isolate No. 115 with sugars and sugar
alcohols as sole and supplementary carbon sources 36
8 Growth of isolate No. 115 on short chain alcohols
and acids when added to basal media 37
9 Values showing GLC retention times of selected
esters on a 15 percent EGS column at 75°C U9
10 Values indicating the shortest length of time
required for ester detection in culture systems
compared to acid catalyzed ester formation in the
absence of culture ^9
11 Ester production in culture supernatant 55
12 Summary of immunodiffusion reactions 57
13 Summary of deoxyribonucleic acid (DNA) density and
base ratio (G/C) analysis 60
lh Values showing initial concentration of metallic ion
in solution (yg/ml) and concentration after shaking for
18 hours in presence of Z. ramigera 115 and I-16-M 8l
15 Gross uptake of metallic ions after l8-hour contact
with pre-grown Z. ramjgera 115 cell-floes 83
16 Gross uptake of various combinations of metallic ions
(two cations) with pre-grown Z. ramigera 115 cell-floes 8U
vi
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TABLES(continued)
No. Page
1? Gross uptake of various combinations of metallie ions
(three cations) with pre-grown Z. ramlgera 115 cell-
floes 87
IB Values (mg/L) showing difference in concentration of
selected cations present in pH 3.0 acid mine water
before and after contact with cell-floe 99
19 Values showing (A) concentration of Fe"*"1"1" and Cl~
(FeClg) adsorbed by cell-floe, and by cell-floe after
washing with (B) anyone of three different anions 101
20 Iron values (mg/L) showing adsorption of Fe from either
pH 2.8 acid mine water or pH 2.8 mine water which was
supplemented with FeCl3 102
21 Values (mg/L) showing uptake of mine water ions by 50 mg
purified polymer per 50 ml flask 103
vii
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FIGURES
No. Page
1 Flow diagram for ester concentration and identification 21
2 Flow diagram showing procedure used to examine isolate
No. 115 culture media for ester production 22
3 Flow diagram showing procedure used to examine isolate
No. 115 cells for ester production 23
k Flow diagram outlining procedure for extraction and
isolation of polymer 27
5 Colonies of Zoogloea raim'gera isolates 115 and I-16-M
after cultivation on TGE agar at 28°C 39
6 Photomicrographs of isolate No. 115 cells showing
capsules resulting in formation of finger-like projec-
tions and zoogloeal slime 40
7 Photomicrographs of (a) isolate No. I-16-M and (b) C-3
showing floes which lack an observable capsular or slime
matrix 41
8 Relative growth of isolate 115 versus substrate concen-
tration in presence of 2% ethyl alcohol 45
9 Concentration of arginine or alanine remaining in
solution after removal of cell floe, versus time 45
10 Infrared spectra of : (A) alcohol extract of Aquacide
concentrated culture supernatant, (b) ethyl butyrate 48
11 Gas-liquid-chromatograph (GLC) of culture supernatant
in presence and absence of ethyl alcohol 51
12 GLC of Aquacide-concehtrated culture supernatant in
presence and absence of ethyl alcohol 52
13 GLC of ether extract of culture supernatant in presence
and absence of ethyl butyrate ^3
Ik pH changes in ethyl alcohol supplemented and non-
supplemented culture media during growth of Z. ramigera
115 54
viii
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FIGURES(continued)
No.
15 Photographs of typical ijnmunodiffusion reactions between
rabbit antisera to floe-formers and trichloroacetic acid
soluble antigens from floe -.formers 59
16 Zoogloeal slime matrix showing interfaces between
individual packets of cells 62
17 Electron micrograph of frozen etched Z. ramigera 115
floes, (l) Lower magnification showing entire floe
packet. (2) Higher magnification showing cell embedded
within matrix 63
18 18-1. Frozen etched Z. ramigera 115 cells showing polymer
strands adhering to individual cells. 18-2 and l8-3«
Negatively stained exocellular polymer strands 64
19 Thin section of Zoogloea 115 floe which contains cells
embedded within a matrix composed of polymer strands 66
20 Purified strands of matrix polymer isolated from Z.
ramigera 115 cells 67
21 Infrared spectrum of purified polymer from Z. rarnlgera
115 cells 68
22 Shadow cast preparations of Z. ramigera I-16-M cells 69
23 Frozen-etched preparations of Z. ramigera I-16-M cells 71
2h Poly-beta-hydroxybutyric acid (PHB) granules obtained
from Z_. ramigera 115 (2^-1) and PHB in Z. ramigera
I-16-M (^2) 72
25 Z_. ramigera I-16-M floes photographed under brightfield
and -ultraviolet illumination after staining with fluores-
cent dye 73
26 Electron micrographs of carbon replicas and frozen
etched specimens of isolate C-3 74
27 Electron micrographs of shadow cast preparations of
isolates (l) Z. filipendula P-8-U, (2) P. denitrificans
P-95-5, (3) isolate C-22-H and (U) Z. ramigera Z-SC-3» 76
28 Electron micrograph freeze etch preparation of a Bacillus
isolate showing cells enmeshed in a fibrillar polymer
network. 37,000 X magnification 79
ix
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FIGURES (continued-)
No.
29 Electron micrograph of a carbon replica of a surface
pellicle of isolate C-3 80
30 Accumulation of Zn+2 by Z. ramigera 115 during growth 90
31 Accumulation of Zn+2 by Z. ramigera I-16-M during growth 92
32 Accumulation of Zn65 by h day culture of 115 cells 93
33 Chemical oxygen demand of medium containing Z. ramigera
115 over a lU day period 94
3^ Accumulation of Zn65 plus 10 ug/ml carrier Zn by a
k day culture of 115 cells 96
35 Interrelationship between the zoogloeal matrix and Zn65
adsorption vs time of Zn addition 97
36 Uptake of Zn+2 by isolated polymer (crude extract) 98
37 Curves showing aldrin adsorption by Gram (+) and G (-)
bacterial floes
38 Curves showing aldrin found in two different contem-
porary sediments and additional aldrin adsorbed by 107
the sediments
39 Scanning electron micrograph of flocculent contemporary
sediment from Lake Erie. 2000 X 109
kO Flask of culture medium illustrating gel formation due
to polymer synthesis
Ul Curves showing viscosity of both whole culture and super-
natant, available sugar and 1KB accumulation vs time
U2 Curves showing viscosity of both whole culture and super-
natant, available sugar and total DNA vs time
k3 Curves showing starch hydrolysis, change in viscosity
and loss of total carbohydrate in culture vs time 114
hk Flow diagram of a treatment plant showing concentration
of metal ions at various locations
^5 Flow diagram of a treatment plant showing concentration
of metal ions at various locations
x
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FIGURES(continued)
No.
h6 Electron micrograph of a shadow cast preparation of Z.
ramigera I-16-M showing adsorption of insoluble mineral
particles to extracellular fibrils 122
1+7 Photomicrograph of the blue green alga Microcystis showing
bacteria embedded within the extracellular slime layer 122
kQ Photomicrograph of an unstained wet mount of a naturally
occurring bacterial-algal floe to which a suspension of
insoluble fluorescent inorganic particles had been added
and then photographed under ultraviolet illumination 123
^9 Phase contrast photograph of the identical field shown
in Fig. hQ 123
50 Electron micrograph of a carbon replica of contemporary
sediment from Lake Erie 125
51 Schematic illustration of the interactions among dis-
solved organic and inorganic pollutants with suspended
microparticles and polymers synthesized by microorganisms 125
xi
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ACKNDWLEDOffiNTS
Contributions of the following individuals during the course of this
investigation is acloiowledged:
Drs. J. I. Frea, R. M. Pfister and F. W. Chorpenning, Depart-
ment of Microbiology, The Ohio State University.
Dr. K. S. Shumate, Department of Civil Engineering, The Ohio
State University.
Dr. C. C. Remsen, Woods Hole Oceanographic Institution, Woods
Hole, Massachusetts.
Dr. R. Marchessault, Department of Chemistry, University of
Montreal.
Former Graduate Students, Department of Microbiology, The Ohio State
University:
Dr. Barry A. Friedman
Dr. Gayle H. Joyce
Mrs. Alice B. Parsons
Mr. Walter Leshniowsky
Mrs. Pamela H. Griffith
Technical assistants:
Mrs. Faith Reilly, James Staber, Karen Nirady, Mrs. Jacqueline
Humpleby, David Schmidt
Use of the facilities at the Water Resources Center, The Ohio State
University.
Support of the Environmental Protection Agency (EPA), Water Quality
Office, U. S. Department of the Interior.
Cecil W. Chambers, Project Officer, EPA.
xii
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SECTION I
CONCLUSIONS
The conclusions put forth are based upon the data and observations
available from the limited number of organisms studied during the
course of this investigation in addition to information available in
the general literature. The general nature of some of the data remains
to be established.
1. Bacterial floe-formation is related to the synthesis and presence
of extracellular polymer fibrillar strands around individual cells.
All floe-formers studied have been shown to produce extracellular
polymer fibrils.
2. The chemical composition of extracellular polymer varies with
bacterial species. The chemical composition and polymeric linkage of
monometric units determines the physical and chemical properties of
individual polymers and, therefore, determines their behavior in solu-
tion. That is, some polymers are more soluble than others, some react
with specific ions to form complexes which readily settle from suspen-
sion, whereas others do not.
3. Excess carbon substrates are converted to extracellular polymer by
the floe-formers. Polymer synthesis can be manipulated nutritionally.
The polymer may be produced to such an extent and be of such a physical-
chemical nature that it is recognized as a loose slime rather than a
well-defined capsule around the cells.
k. Some polymers bind water to a greater extent than others. The more
soluble extracellular polymers which become detached from cells and
remain in colloidal suspension will result in increased viscosity of
the surrounding medium when produced extensively by the cells. In
waste treatment processes, this would have the net effect of reducing
flocculation although it results from the factor that causes floccula-
tion. This situation could be reversed by lowering the C:N ratio of
input nutrients (sewage) or by addition of a chemical that will complex
with the polymer strands.
5. Polymer can be produced in large amounts and this can be exploited
as a means of removal of chemicals from solution providing the chemi-
cals are of a type that form a complex with the polymer strands. There
appears to be a high potential for removal of many cations from solu-
tion (e.g., transition and heavy metal ions) by this process. Second-
ary associations (e.g., anlons to the cation-polymer complex) will
allow extension of this procedure.
6. All of the polymers from the gram negative bacterial isolates
studied are polysaccharides. These polymers have a high affinity
for binding water. The polymer and associated water is a gelatinous
mass from which the water is difficult to remove.
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7. Presumably the water is organize4 in polarized ordered multilayers
surrounding the polymer strands. This bound water appears to exchange
with many metal ions. In so doing, a metal ion-polymer complex is formed
which will precipitate (flocculate) from colloidal suspension and sepa-
rate from the bulk solution.
8. It is postulated but not proven that water can be exchanged in two
ways: (l) By chemicals in solution that have a greater affinity than
the polymer for polarizing and orienting water in multilayers around
them. In this way water would disorient from the polymer in favor of
the ion or other chemical in solution. (2) By chemicals in solution
that have a greater affinity for forming an association with the polymer
structure than water does. This would result in a complex formation via
exchange with bound water and would be similar to a polyelectrolyte or
ion exchange process. In either case the physical properties of the
polymer colloid would be altered and depending upon the ion or chemical
associating with it, either flocculation or increased solubility could
result.
9. In the case of a polymer matrix which surrounds cells, metal ions
accumulate with the polymer and do not reach the cell membrane surface.
This explains the high tolerance of such cells for ions that are normally
toxic to cells.
10. With regard to $9 above we have also shown that organic nutrients
(e.g., amino acids) are similarly adsorbed from solution and are oxidized
by the cells later in time. This explains the high rate of BOD removal
observed for floe-forming bacteria and also indicates that such cells
can accumulate nutrients from an extremely dilute nutritional environ-
ment.
11. Insoluble particles such as clay, other bacteria, detritus, etc.
can adsorb to polymer strands (See Fig. 1*6). This is referred to as
formation of a conglomerate floe. It is a moot question as to whether
particulates adsorb to polymer or polymer adsorbs to particle. Therefore,
if the particle is of sufficient size the overall appearance will be
adsorption of polymer to a surface or interface. In the latter case the
polymer could be surrounding cells and then be attached to any suitable
surface. For example, zoogloeal masses adhere to limestone surfaces in
a trickling filter, encapsulated cells adhere to the walls of a flask or
other container, polymer producing cells adhere to teeth and in general
polymers collect at interfaces.
12. To a large extent the adsorption, concentration and accumulation of
chemicals from solution via the mechanisms described are physio-chemical
processes that are relatively non-specific by comparison to the high
degree of specificity usually found in biological metabolic reactions.
13. Flocculation and floe-forming processes based upon the physio-chemical"
biological interactions described are the same in waste treatment systems
as they are in lakes. The differences between activated sludge removal of
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oxygen demand and lake eutrophication is a matter of degree and complexity
in so far as flocculation and its effects are concerned.
l^J-. The gram negative floe-formers studied in this report all appear to
be related taxonomically. That is, they cluster when analysed using
numerical computer techniques and they share either identical or cross-
reacting TCA soluble antigens. When compared to isolates obtained from
other culture collections the organisms appear to be related to organisms
which various investigators have identified as Zoogloea, Pseudomonas ,
Acetomonas, Acetobacter and Gluconobacter. Of course, many floe-formers
have been observed which are known on the basis of other traits to belong
to a variety of genera other than those named, including gram positive
organisms.
15. Microbial production of polymers having ion adsorption and exchange
capacities has potential for use in treating industrial waste water -
particularly water contaminated with metal ions or radioactive minerals.
16. The reverse of the above conclusion (#15) has potential for control-
ling the chemical and physical properties of slime and biological floe
in situations where slime is undesirable.
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SECTION II
RECOMMENDATIONS
1. Pilot scale studies should be made for the purpose of scaling-up the
polymer production-metal ion adsorption-flocculation process as a poten-
tial means of removing organic compounds and ions on a selective basis.
For example, toxic metal ions or radioactive ions could be stripped from
waste processing water prior to having it dumped into municipal sewage
systems or other receiving water.
2. Adsorption to floe-former polymers of chemicals other than those
examined have far reaching ramifications and should be studied. For
example, enzyme adsorption has considerable potential with regard to
sewage digestion and solid waste treatment. Health related implications
in the area of tooth decay and diseases caused by encapsulated organisms
(pneumonia, etc.) are undoubtedly related to the general phenomena
described and should be investigated in that regard. Also the basic bio-
logical processes of mineral deposition such as in bone, teeth, shell-
fish, etc. are likely related to adsorption to polymers synthesized by
cells and should be examined.
3. The high bound water content of activated sludge is a major problem
and deterrant to its disposal. Means of separation of bound water from
polymer in sludge should be studied and approaches to the problem are
suggested by the conclusions of this report.
k. The generality of the conclusion of this report should be further
established particularly as related to mixed cultures and environmental
conditions (e.g., temperature and pH extremes) where practical abatement
processes could be developed.
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SECTION III
INTRODUCTION
The process of aerobic biological waste treatment is largely dependent
upon two biological events: (A) Conversion of dissolved or suspended
organic substances to microbial biomass via metabolic reactions. Since
the organic substances are in a somewhat reduced chemical state, and
therefore oxidizable, removal by metabolic conversion to cell substance
represents biochemical oxygen demand (BOD) removal once the cells have
been physically removed from the system. (B) The aggregation or floccu-
lation of the biomass thus formed, results in settling and thereby pro-
vides a means for mechanically separating cells from supernatant (or in
sanitary engineering terms, separating sludge from liquor). To a large
extent, sewage or other organic wastes can be considered as microbial
nutrients which are either in solution or suspended macro-molecules that
are susceptible to attack by the hydrolytic enzymes produced by microbes.
Flocculation affords a means of physically separating suspended particu-
lates, including microorganisms and adsorbed substances, from the water
phase. The suspended particulates found in waste water and sewage consist
of colloidal dispersions of a variety of bacteria and other microorganisms
in addition to non-living organic and inorganic substances. Flocculation
is the aggregation or clumping of dispersed particles resulting in a floe
which will settle from suspension upon standing, thereby leaving a clear
supernatant.
The role of bacteria as the agents primarily responsible for flocculation
as well as oxidation of reduced organic wastes in sewage treatment has
long been recognized. Floe-formation appears to be related to the pres-
ence of capsular and/or slime polymers which are synthesized by bacteria
and other microorganisms during the process of oxidation. In many
instances the individual bacterial cells are embedded within a slime or
capsular matrix which is often called a zoogloeal matrix. Some of the
bacteria that produce rather characteristic zoogloeal matrices have been
taxonomically classified as Zoogloea. Other bacteria which characteristi-
cally form floes but differ in that they do not produce a typical zoo-
gloeal matrix, have been identified as belonging to one of several other
bacterial genera.
The biological phenomena described above as being basic to the success
of aerobic BOD removal are complicated by adsorption on suspended parti-
cles of inorganics as well as organics that are not readily oxidized.
Adsorption is a physical-chemical phenomenon but the biological forma-
tion of adsorption sites on sludge floe also relegates this to a consider-
ation which overlaps biology and we will consider this aspect in more
detail.
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During the course of this investigation to examine the role of floc-
forming microorganisms as biological agents for removal of BOD and toxic
chemicals (metal ions, pesticides, etc.) from water, it became necessary
to follow several divergent lines of research. It was considered essen-
tial to study (a) the process of flocculation; (b) the means or mecha-
nisms by which these organisms exert a high rate of BOD removal; (c) mecha-
nisms by which the bacteria either oxidize chemicals in solution or other-
wise remove non-oxidized chemicals via complexing or absorption reactions;
(d) examine in detail the structure and function of zoogloeal matrix
material for its unique role in a, b, and c above; (e) study the process
by which ceUs produce the zoogloeal matrix because of its potential as
an abatement process for removal of selected chemicals from contaminated
water and (f) attempt to clarify the confusing taxonomic status of the
floe-forming bacteria in general and Zoogloea species particularly.
The general pattern of this report is to consider each of the above
objectives (a through f) as separate phases which are discussed in con-
text of pertinent related phases.
Research was initiated for the purpose of examining the functional role
of the gelatinous matrix which surrounds, and is characteristic of the
bacteria classified as belonging to the genus Zoogloea (10). No Zoogloea
bacteria were available from culture collections or from laboratories
which ha'd reported on Zoogloea at the onset of this study. Bacteria were
isolated from water samples and a strain was characterized and identified
as Zoogloea according to published descriptions (9, 10, lU, ^69 100).
During our investigation into the nature of the zoogleal matrix a descrip-
tion of Zoogloea was published which stated that Zoogloea species do not
possess a capsule or recognizable slime layer. On the basis of this
description, the authors requested recognition of their cultures as the
neotype of Z. ra-nrigera Itzigsohn (29). This report necessitated a reap-
praisal of the identification of our isolates and prompted a more thorough
study of the taxonomic status of Zoogloea in general. Reappraisal of cell
morphology, particularly with reference to capsule or zoogleal matrix
formation, led to an examination of literature pertaining to zoogleal-
fonning bacteria which are not generally recognized as belonging to the
genus Zoogloea.
One objective of this report is to establish that an organism identified
as Z. raim'gera, based upon published descriptions, does possess a clearly
recognizable gelatinous matrix. However, there appears to be no reason
not to classify the same organism as a Siderocapsa species based upon
published descriptions of this genus. The relationship of Zoogloea to
gelatinous matrix-producing genera of the Siderocapsaceae is therefore
considered. Further, the formation of a zoogleal matrix by a bacterial
strain is not synonomous with floe formation since many species of bac-
teria are known to flocculate, but without synthesizing a gelatinous
matrix (80)-
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Zoogloea ramigera is a gram-negative rod which appears to be a pseudo-
monad. It is characterized on the basis of its flocculent growth habit
(ik, 2.6, 33, UO, 93, 98) and a unique extracellular matrix which may be
analogous to a capsule (l^j ^0> 93 > 9*0 • The isolate Z. ramigera 115
has been described as producing a zoogleal or capsular matrix, whereas
another isolate, Z. ramigera I-16-M, has been described as having no
matrix. Comparisons between these two isolates regarding flocculation
and biochemical characteristics are reported in addition to comparisons
to several other floe-forming pseudomonads (^0). The extracellular
zoogleal matrix has previously been examined for the purpose of elucida
ting its functional properties. The capacity of the extracellular
polymer for concentrating metal ions from solution has been reported,
and some of the ecological implications have been discussed (Ul).
The structure and composition of the zoogloeal matrix is considered in
relationship to metabolic activities of the organism, which has been
reported to possess an extremely active oxidative metabolism in the
natural habitat (lU, 16, 98) .
Several bacteria have been isolated in this laboratory and elsewhere on
the basis of their flocculent growth habit. Floc-formlng bacteria grow
primarily as clumps of large numbers of cells which may be surrounded
by an extracellular polymer matrix (91 )« Because floes settle out of
suspension in the absence of agitation the medium rarely becomes turbid.
A distinction is made in this paper between "floe -format ion" which implies
growth in a clumped arrangement s and "flocculation11 which implies aggre-
gation from a turbid suspension after the cells have grown. This latter
phenomenon is often referred to in the literature as "flocculation" (11,
12, 13, 23, 7^, 83, 91) and is often an effect caused by the addition of
chemical agents (e.g., blood sera, bentonite, polyvalent ions). On a
physical-chemical basis the causes of flocculation and floe -format ion may
be similar with the differences being based on the origin and time involve-
ment of the substance causing the clumping phenomenon.
Floe -forming bacteria are indigenous to natural water and the flocculent
growth habit is exploited in biological waste treatment processes. Bac-
terial flocculation has been discussed by Crabtree et al. (28) and an
explanation of the flocculation based upon induction by poly-beta-
hydroxybutyric acid (1KB) storage granules has been proposed (26) .
Tenney and Stumm (91) and Busch and Stumm (13) have described bioflocu-
lation as "an agglomeration of cells resulting from specific adsorption
of polymer segments and from bridging of polymers between cells." These
authors presented chemical data based upon the ability of synthetic
polyelectrolytes to cause aggregation of bacteria. They were able to
show that polymers extracted from Aerobacter aerogenes behaved in the
same manner as synthetic polyelectrolytes .
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The Zoogloea ramigera strain 115 was shown to be composed of polysaccha-
ride susceptible to cellulase and therefore believed to contain B-l-^,
linkages (k2). The polymer also possesses properties of a polyelectro-
lyte (^0), as it binds high concentrations of metallic ions. Although
the polymer is not readily observable around all Zoogloea isolates by
techniques such as capsule stains, observation using electron microscopy
revealed the presence of extracellular fibrils. These observations led
us to examine several floe-forming bacterial isolates for the presence
of extracellular polymer and to present microscopic evidence involving
extracellular polymers as a cause of bacterial flocculation.
Extracellular matrix synthesis is, however, related to growth conditions
and is, therefore, not an adequate taxonomic criterion (28, kQ, 93)•
The fibrillar nature of the extracellular matrix of Z. rarnigera 115 has
been established (^2). Polymer fibrils that resemble cellulose have been
observed around additional Zoogloea isolates as well as non-Zoogloea
floe-forming bacteria (^3). This has led to the consideration of possi-
ble taxonomic relationships among the so-called floe-forming Pseudo-
monadaceae and Zoogloea to recognized species of Pseudomonas and Acetobacter,
There are few reports concerning antigenic relationships among the
Pseudomonadaceae other than for the genus Pseudomonas. An exception is
the work of Mclntosh (63) in which an antigenic analysis of various
Acetobacter species was made using agglutination and tube precipitation
tests. He reported that the A. suboxydans strains constituted a distin-
guishable serologic group and that A. rancens strains also exhibited
serologic relationships, but were unrelated to A. suboxydans. A. oxydans,
A. xylinum, and A. melanogenin were not included in the study.
This report is intended to demonstrate and clarify: (i) antigenic rela-
tionships among similar isolates which produce cellulase susceptible
extracellular polymers, (ii) to compare them to type species of Pseudomonas
and Acetobacter, and (iii) to establish the relationship of isolates clas-
sified as Zoogloea to Pseudomonas and Acetobacter.
Antigenic relationships of the four isolates were examined by innnuno-
diffusion and were compared to three cultures obtained from American
Type Culture Collection. The type cultures were selected on the basis
of similarity of substrate range, particularly alcohol metabolism, and
because Acetobacter is the only pseudomonad reported to synthesize cellu-
lose which is of course a cellulase susceptible extracellular polymer.
Biological waste treatment processes convert reduced organic substances
to microbial cell mass. The energy required to drive this synthetic process
is derived from oxidative reactions catalyzed by the enzyme systems of the
microorganisms. Once the organic material has been converted to microbial
cells, biological flocculation becomes an essential part of the process
because it forms the basis for removal of partially oxidized material from
the water. Relatively little is known with regard to the role of dissolved
minerals in the treatment process, particularly in relation to growth -of
microorganisms. Current opinion is that mineral content as well as organic
8
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content of treated waste plays an important role in stimulating growth
of algae and other plant forms in receiving waters (36, 39)•
Our interest in Zoogloea species concerns the ability of the organism
to concentrate and accumulate dissolved chemicals from solution. We
are also attempting to elucidate the functional role of the gelatinous
matrix in the metabolic activities of the organism. This study is stimu-
lated by potential application in the following areas: (i) as a means
for selective removal of minerals and/or organics by waste treatment
processes resulting in a retarding influence on the eutrophication pro-
cess in receiving waters, (ii) recovery of isotope contaminants from
aquatic environments, (iii) basic information concerning nutritional
requirements and environmental influences on activity of the organisms
will allow more efficient design of specialized aerobic treatment facili-
ties.
The gelatinous matrix producing Zoogloea ramigera strain 115 (^-0) and
the nongelatinous matrix producing strain I-lo-M of Crabtree et al. (26,
28) were studied with respect to metal ion uptake.
After adsorption of individual ion pairs from solutions was examined,
an evaluation of a complex mixture of ions from a natural source was
studied. Samples of acid mine drainage were selected for this purpose.
Acid mine drainage is characterized by high concentrations of dissolved
iron, sulfate and hydrogen ions, as well as various other ions in lower
concentrations. It is the adverse influence of these ions both in
drainage streams and in receiving water that elicits the water pollu-
tional stigma so often associated with mining operations.
My colleagues and I have argued that the most sensible approach to
control of mine drainage pollution would be to prevent its formation at
the source. One specific suggestion was to selectively inhibit the
acidophilic autotrophic bacteria (Thiobacillus-Ferrobacillus group).
These organisms are responsible for production of much of the sulfate
and ferric iron ions via metabolic oxidation of pyrite and related
minerals. They are indirectly responsible for a considerable amount
of acid formation because the ferric ion they produce, reacts with water
yielding "yellowboy" and acid (H4").
Treatment of the problem, once formed, is then a "catch up" procedure
and acid pollution abatement would be a continuous effort. Although
treatment is not an ultimate solution to the problem, situations do
arise where immediate action is demanded and practical means of preven-
tion are not yet available. We take the viewpoint that treatment is
essential while adequate preventative measures are being sought, and
have suggested the utilization of sulfate reducing bacteria as having
potential in this regard. Anaerobic sulfate reducing bacteria in the
De sulfovibrio and De sulfotomaculum genera have the demonstrated capacity
to reduce sulfate to sulfide with concommitant increase in pH and removal
of iron ions by precipitation as black FeS.
-------
The present suggestion represents another potential microbiological
treatment process for removal of mine water ions. The data herein
pertain to removal of ions found in acid mine drainage by adsorption
to extracellular polymers that are synthesized by microorganisms under
their natural growth conditions. In this context I would like to
emphasize that this is analogous to the processes now in use for treat-
ment of municipal and domestic wastes. Virtually all such processes in
current use are biological processes which depend upon the metabolic
activity of microorganisms. The point to be made is that much of the
engineering technology is already available and could be extrapolated
for use in abatement of acid mine drainage pollution.
Many chlorinated hydrocarbon insecticides have been isolated from sur-
face waters, usually in concentrations of less than 1 p,g/liter. Sig-
nificance of the presence of pesticides in low concentrations has been
established (25), for example, with reference to accumulation in shell-
fish and also in regard to chronic synergistic influences on fish.
Certain questions arise. For example, how do highly insoluble chlorin-
ated hydrocarbons enter the aquatic system and once there, how are they
transported? Organic pesticides can enter the ground and surface
waters in a number of ways: by direct application for control of aquatic
weeds, trash fish, and aquatic insects; from discharge of industrial
waste water; by runoff from agricultural-land; by drift from aerial and
land applications; from clean-up of equipment used for pesticide appli-
cation; and finally, "by accident.
Our interest lies in the fate of these chemicals once in the water
column, and particularly, in their adsorption to silt and floe-forming
bacteria which form contemporary sediment in lakes.
In this context, the adsorption of the chlorinated hydrocarbon pesti-
cide, aldrin, by floe-forming bacteria and lake silt was selected as
an experimental system, although any one of a number of other organic
pesticides could have been used.
A preliminary study of aerobic heterotropic bacteria isolated from Lake
Erie indicated that of thirty-eight bacteria isolated and examined, four-
teen formed floes in at least one of the six different growth media
tested, one of which was sterile lake water. Two of the isolates which
formed floes in sterile lake water were selected for further study with
regard to their ability to concentrate and accumulate the pesticide
aldrin from solution. One bacterium was an orange-red pigmented gram
negative (G-) pseudomonad rod. The other was a gram positive (G+)
Bacillus species.
Once it had been demonstrated that floe-forming bacteria were
ubiquitous in nature and that the polymer produced by the floe-formers
would adsorb a wide variety of chemical substances (anions, cations,
soluble and insoluble organics, pesticides, inorganic and organic in-
soluble particulates), it was of interest to investigate the capacity
10
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of cells for producing extracellular polymer. Further investigations
of polymer formation, organic oxidation and the influence of growth
conditions on polymer production ensued, with the ultimate objective
of evaluating the potential for large scale polymer production and
its use for water treatment on a large scale.
11
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SECTION IV
MATERIALS AM) METHODS
Organisms
Zoogloea ramigera 115 and Pseudomonas species C-3 were isolated from a
trickling filter at this laboratory and have been previously described
(UO, te, 1*3). Z. filipendula P-8-If, Z. ramigera I-16-M, P. denitrificans
P-95-5 and an unidentified gram negative rod C-22-4 were also isolated
from sewage by Crab tree et al. (ifO) and considered elsewhere (UO, U2,
^3). P. denitrificans (ATCC 1386?), Acetobacter oxydans (ATCC 6^33),
and A. suboxydans (ATCC 621) were obtained from the American Type Culture
Collection. Z_. ramigera Z-SC-38 was isolated by Ganapati et al. (Mf).
Isolation of Organisms
Isolations were made from limestone covered with zoogleal masses and
effluents taken from organically polluted water. Material on the stone
was scraped off and placed into 0.05$ proteose-peptone yeast extract
broth (PPYE, Difco). The gelatinous material was disrupted by exposing
small clumps to ultrasound (Branson, 20Kc output) in sterile PPYE until
the clumps dispersed,, Samples were serially diluted in PPYE and incu-
bated at 28°C. After three days the highest dilutions containing growth
(10~7 to 10-10) were streaked onto PPYE agar and Tryptone Glucose Extract
Agar (TGE, Difco). When sufficient growth occurred, colonies of differ-
ing morphological types were transferred to PPYE in duplicate. One tube
was placed in an incubator at 28°C and the other was placed on a rotary
shaker (60 rev/min) at 2h° ± 1°C. Incubation was continued until growth
was observed. Those tubes containing a pellicle (stationary) or a floe
(shaken) were selected and the entire isolation procedure was repeated
until pure cultures were obtained.
Characterization of Organisms
Cells were inoculated into 100 ml of 0.05$ PPYE or 0.5$ Trypticase Soy
Broth (BBL) and grown on a rotary shaker at 2V C ± 1°C for U8 hours.
They were washed twice with 0.3M phosphate buffer and resuspended in
10 ml of buffer.
Casein Hydrolysate (0.2$ enzymatic, Difco) containing 16 |ag/ml brom thymol
blue indicator served as the basal medium for carbohydrate utilization
studies. Carbohydrates were added to the basal medium to give either 1$
(w/v) solutions or as differential discs (l disc/7 ml, Difco). Sugars
other than discs were sterilized by autoclave. Occasionally a slight
acid reaction was detected as the result of autoclaving; in these cases
the medium was sterilized by membrane filtration. Alcohols were prepared
in 1$ concentrations. Crystal violet was added to TGE to give a final
concentration of 0.001$ for use as a differential growth medium. All
other media were prepared according to standard methods (8?)• Tubes were
12
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inoculated with 0.1 ml of a cell suspension and results were read after
2*1, lj-8, and 120 hours.
Growth of three isolates designed as 115, C-3, and I-16-M was examined
in various defined growth media for the purpose of comparing flocculation,
zoogloea matrix formation, and growth. The media examined were: (A) the
arginine medium of Crabtree et al. (2?). This medium was varied by add-
ing (A.) folic acid, 10 mug/ml; or (AQ} folic acid, 10 mjig/ml plus glucose,
0.5$, (B) the ammonium nitrate medium of Crabtree (Ha.D. thesis, Univ. of
Wisconsin, Madison, 1966) NH^MOa, 0.1$; MgS04-7HOH, 0.02$; KsHKU, 0.2$;
KH2K)4, 0.1$; and glucose, 0.5$. This medium was varied by adding either
(BL) vitamin B^, 1.5 mug/ml; or ('B2) B-^, 1.5 mug/ml plus folic acid,
10 mug/ml plus biotin, 2.0 m|ig/ml. (c) the casamino acid medium of Crab-
tree (Hi.D. thesis, Univ. of Wisconsin, Madison, 1966), which contained
vitamin free casamino acids 0.1$ (Difco); MgSOA'THOH, 0.02$; K^HP04, 0.2$;
KH2K>4, 0.1$; Bj.2, 1.5 m|ag/ml; biotin, 2.0 mug/ml. This medium was varied
by adding either (c^) folic acid, 10 mjig/ml or (C2) folic acid, 10 mug/ml
plus glucose, 0.5$.
Because of time and manpower constraints all isolates examined could not
be studied in equal detail within the scope of the experimental objectives.
Therefore, greater attention was given to Zoogloea rarrrigera H5 than to
other isolates due to interest in the uniqueness of the organism as a
zoogloeal matrix producer. Z_. ramlgera 115, in addition to a closely
related isolate, Z. ramigera 1, were further characterized biochemically
and nutritionally in an attempt to assess their role as agents for BOD
removal in waste treatment processes.
The isolates have the following general descriptions after 72 hours culti-
vation at 25°C on PPYE medium which contained 0.001$ crystal violet
(either broth or 1.5$ agar).
Isolate 1: Gram-negative, freely motile rods, 0.5|j, long; sudanophilic
granules present. Colonies were straw-colored and leathery
to needle touch, round with an entire margin. No ridges or
convolutions were present. Crystal violet was decolorized.
Isolate 115: Gram-negative rods, Ijj. long; sudanophilic granules present;
cells held together in a floe or matrix but swam away from
the finger-like projections of the matrix. Colonies were
circular with a slightly scalloped or convoluted margin,
leathery to the touch and difficult to break up. Crystal
violet was decolorized.
Culture Conditions
The following basal growth media were used for studies on substrate uti-
lization in addition to standard differential media (8?).
13
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Basal medium I (Dugan and Lundgren, I960)
KH2P04 2.5 g CuS04-5H20 0.1 g
K2HP04 1.25 g FeS04-?H20 0.1 g
MgS04 1.0 g MnS04«H20 0.1 g
NaeMo04 0.1 g ZnS04-7H20 0.1 g
Boric Acid1 0.1 g CaCl2'2H20 0.1 g
Co(N03)2-6H20 0.1 g
Double distilled deionized H20 1000.0 ml
Basal medium II (Crabtree et al., 1966)
MgS04-7H20 0.2 g
KgHKU 2.0 g
KH2P04 1.0 g
Distilled H20 1000.0 ml
Basal medium III (for primary carbon sources)
Casamino acids, vitamin free 3.0 g
Vitamin B^ 1.5 x 10~4 g
Basal medium I 1000.0 ml
Basal medium IV (for primary carbon sources)
Yeast extract 0.5 g
Basal medium III minus B12 1000.0 ml
Basal medium V (for primary carbon sources)
Biotin 0.01 g
Folic acid 0.01 g
Thiamine 1.0 g
Basal medium III minus B12 1000.0 ml
Basal medium VI (for sole^ carbon sources)
(]H4)2S04 0.5 g
Biotin 0.01 g
Folic acid 0.01 g
Thiamine 1.0 g
Basal medium I 1000.0 ml
Proteose Peptone-Yeast Extract (PFYE) medium
Proteose Peptone 0.5 g
Yeast Extract 0.5 g
Distilled H20 1000.0 ml
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Chit in and Collagen Media
Basal medium I, supplemented with yeast extract (5 mg/ml), was employed
in the examination of isolates 1 and 115 for the presence of extracellu-
lar chitinase and collagenase. Chitin or collagen, in 10$ (w/v) concen-
trations, were dispersed in distilled water in a Waring Blendor. These
suspensions were sterilized separately from the basal medium by auto-
claving at 121°C for 10 min. They were then added aseptically to the
liquid medium for Erlenmeyer flask shake cultures (New Brunswick platform
shaker, 160 rpm) and to solidified plating medium, to give a final con-
centration of 1 mg/ml.
Carbon and Nitrogen Sources
Basal medium II was employed in conjunction with various nitrogen and
carbon sources, supplemented with the following growth factors: (A)
vitamin Bi2 (1-5 x 10-7 (ig/ml); (B) folic acid (0.01 |ig/ml), biotin
(0.01 ng/ml), thiamine (1.0 ng/ml); (C) Yeast Extract (10 ng/ml). (This
concentration did not supply sufficient carbon and nitrogen to support
growth.) pH was adjusted to 7-0 using either 2N HC1 or 2N KOH.
Ammonium salts. NK^NOs, (NH4)2S04, and NH4C1 were tested as nitrogen
sources in basal media I and II, at 0.5 mg/ml concentration. The three
growth factor systems mentioned above were studied using the following
concentrations of glucose as carbon source: 0.25 mg/ml, 0.5 mg/ml,
1.0 mg/ml, 2.5 mg/ml, 5-0 mg/ml, and 10.0 mg/ml.
Amino acids. The examination of 20 amino acids as sole carbon and nitro-
gen sources was carried out in basal media I and II, with vitamin B^
and yeast extract as growth factors. The basal media were supplemented
with glucose (20 mg/ml) when it was desired to determine the ability of
the organism to use the amino acids as a source of nitrogen only. Ami Tin
acids were supplied in concentrations of 1.5 x lO'^M, 1.5 x IQ-3!!, and
1.5 x 10-^.
These same basal media, both with and without the amino acids, were
supplemented with guanine, cytosine, and adenylic acid, individually
and as a mixture to give a final concentration of ?0 ng of each nitrogen
base per milliliter of medium. Stationary and aeration tube cultures
were used in this study.
Sugars and sugar alcohols. The ability of isolate 115 to utilize numerous
sugars and sugar alcohols as primary and sole carbon sources was examined
in basal media II (containing either arginine (0.5 mg/ml) or (NH4)2S04
(0.5 mg/ml), III, IV, V, and VI. All sugars were filter sterilized
(Millipore, O.ij-5 u pore size) and added to the basal media (50.0 mg/ml)
in Erlenmeyer flasks and incubated at both 2h°C and 30° C on a plitform
rotary shaker. The cultures were examined for changes in pH with a
Corning expanded scale pH meter (Corning Instrument Co., Corning, N.Y.)
at 12-hr intervals for 5 days.
15
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Organic acids and alcohols. Several organic acids and alcohols were
examined as sole and supplementary carbon sources. It was assumed that
concentrated methyl, ethyl, propyl, isobutyl, sec-butyl, n-amyl, dodecyl,
lauryl, and stearyl alcohols would not contain living contaminants, and
the alcohols were added aseptically directly to sterile "basal media.
Formic, acetic, propionic, butyric, alpha-ketobutyric, beta-hydroxbutyric,
caproic, and alpha-ketoglutaric acids were filter sterilized. Capric,
lauric, and stearic acids were autoclaved before addition to the basal
media. The basal media used were I and II, supplemented with arginine
or (KH4)2S04, and PPYE. The final concentrations of acids or alcohols
in the media were 0.5$, 1.0$, and 2.0$ (liquids, v/v; solids, w/v).
The studies were carried out in shaken Erlenmeyer flasks, aeration tubes,
and on agar plates. Homogenous media were not obtained with C12 and
C18 acids and alcohols.
Preparation of Antigens
For the immunization of rabbits, antigens were prepared as follows.
A. oxydans was grown in 0.5$ Tryptone Glucose Extract broth (TGE, Difco)
at 26 C for 2k hours. All other organisms were grown in 0.05$ Trypti-
case Soy Broth (TSB, Difco) under the same conditions. The cells were
washed twice with 0.85$ saline, resuspended in 20 ml of saline, treated
with ultrasound for 1/2 to 3A minute with a Bronwill Biosonik III
Sonicator (Bronwill Co., Rochester, N. Y.) to disrupt the floe and
polymer, and diluted with saline to correspond to a nephelometer tube
calibrated to 3 x 10s cells/ml.
For the test antigens, cultures were grown for two to three days at 26°C
in 12-liter fermenters containing either 0.05$ TSB or 0.5$ TGE. The
cells were collected with a Sorvall RC2B Superspeed Centrifuge, using
the KSB continuous flow apparatus (Ivan Sorvall, Inc., Norwalk, Conn.).
A modification of the trichloroacetic acid TCA extraction method of
Van Eeden (96) was employed to prepare the bacterial antigens used in
serologic tests, the TCA soluble extracts were dialyzed at k°C against
three changes of phosphate-buffered saline (pH 7«3)j instead of a water-
thiomersalate mixture. An extract of 0.005$ TSB was prepared in the
same way, as a control. The extracts were stored at k°C after the addi-
tion of 0.02$ sodium azide, as a preservative.
Gel Diffusion Tests
A modification of Campbell's (17) method was employed. Seventy-five
milliliters of 0.85$ saline and 15 ml of double-distilled water were
added to 0.85 gm of Special Agar Noble (Difco). The mixture was auto-
claved for 15 minutes, allowed to cool to 55°C, and 10 ml of 1:1000
stainless merthiolate (Eli Lilly Co., Indianapolis, Ind.) and 2.5 ml of
0.1$ aqueous trypan blue was added to the cooled agar. Five millimeter
wells were cut 9 mm from center to center in a 3-0 mm agar bed in Petri
dishes, and 0.05 ml of reactant was added to each well. The plates were
incubated at 37°C for 2^-36 hours. Sera were concentrated by a factor
16
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of 2 or 3 with a Diaflo Model 50 ultrafiltration cell containing a DM
10 membrane (Amicon Corp., Lexington, Mass.)- The TCA-extracted antigens
were concentrated by a factor of k or 16 by pervaporation with subsequent
dialysis against buffered saline (pH 7.3). After testing each antigen
with each serum in hexagonal well patterns, all indications of possible
relationships were re-examined separately in adjacent wells. Inconclu-
sive reactions were aided by further concentrating the antisera and
antigens involved.
Nucleic Acid Base Ratio Determination
Deoxyribonucleic acid (DNA) was extracted by a modification of the Marmur
procedure (62). Because of the large amount of polysaccharide present
it was necessary to homogenize the samples in the presence of sodium
lauryl sulfate. Polymer was precipitated by adding isopropyl alcohol in
place of ethyl alcohol during the initial DNA precipitation before pro-
ceeding with the deproteinization step. In many cases it was not neces-
sary to treat with RNase after deproteinization because DNA. was selec-
tively precipitated during early steps in the procedure. It was assumed
that the large amount of polysaccharide adsorbed salts from solution
which thus altered the delicate salt balance that controls precipitation
of the DNA.
DNA base composition ($ G-C) was determined by the buoyant density method
of Shilderkraut et al. (84). The DNA-CsCl solution was centrifuged in a
Sectarian model E analytical ultracentrifuge (AiiD rotor) at 1*0,000 rpm and
23°C. Photographs of the density gradient were taken at k hr. 16 min.
intervals.
Microscopy of Cells
(A) Cultures. Cells were grown in a modified version of Crabtree's (27)
arginine salts medium which contained per 100 ml: arginine«HC1, 0.05 g;
MgS04'7HOH, 0.02 g; KaHPO^ 0.2 g; KH2P04, 0.1 g; glucose, 0.5 gj Bj.2,
1.5 x 10-7 g. Cultures were shaken at 28°C for 72 hours.
Brightfield and ultraviolet microscopy. A 0.1% aqueous solution of Paper
White-BP (a diamine stilbene disulfonic acid dye which has a high affinity
for cellulose; E. I. DuPont de Nemours and Co. Inc., Chicago), was used
to stain 72 hr cultures of Z. ramigera 115 and Z. ramigera. I-16-M in a
manner similar to that described by Harrington and Raper (1*7). The
stained cell floes were examined by both brightfield and ultraviolet
microscopy. Photomicrographs were taken on Kodak Plus-X film. Exposure
times were 5 to 8 sec in visible light and 15-90 sec in ultraviolet
light.
(B) Preparation for electron microscopy. Z. ramigera isolates 115 and
I-16-M were grown in a modification of the~~arginine salts medium of
Crab tree (28) which has the following composition per 100 ml: arginine-HCl,
0.05 g; MgS04-7HOH, 0.02 g; K2HP04j 0.2 g; KH2P04, 0.1 g; glucose, 0.5 g;
17
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!»5 x 10-7 g. Proteose-Peptone yeast extract, 0.05% (PPYE, Difco)
was used to obtain flocculent growth of cultures other than Z. ramigera
115 and Z. ramigera I-16-M. The bacteria were cultured in Erlenmeyer
flasks containing 100 ml of growth medium and were either incubated on
a reciprocal shaker (100 strokes/min) at 28°C until flocculent growth
occurred, or held stationary at 28°C until a pellicle formed. Floes or
pellicles were washed twice with distilled water. In some experiments
this was followed by boiling the cell-floes in 1 N NaOH for 20 min. (?2)
or by allowing them to stand for 2k hours at 28 °C to remove alkali-
soluble material.
(C) Shadow-casting and carbon replicas. The cell-floes were placed on
Formvar coated grids and shadowed with carbon platinum or they were
shadowed and then carbon coated. Carbon replicas were prepared by the
technique of Bradley and Williams (8).
(D) Freeze-etchjng. The floes were centrifuged into a pellet and a
portion of this was placed on a Rrna.11 (3 mm) copper disc, previously
scratched to insure greater adherence. The disc was immediately frozen
in liquid Freon 22 and transferred to liquid nitrogen. The object was
then placed on a pre-cooled (-150°C) table in a Balzers apparatus (model
BA 360 M; Balzers, Principality of Leichtenstein) and frozen-etched as
described by Moor and Muhlethaler (6?) and Moor (66). After freeze-
etching, the discs containing the specimens were removed, warmed to
room temperature, and dipped into distilled water to remove replicas
from specimens. To remove cells which remained attached to replicas,
treatments of 70% sul±uric acid (l hr.), distilled water rinse, Eau
de Javell (a commercial bleaching agent containing 1*4$ NaHOCl in NaOH),
and final distilled water rinses were employed. After the replicas were
picked up on Formvar-coated grids and allowed to dry, they were ready
for examination in the electron microscope.
(E) Negative staining. The cell pellets from centrifuged floes were
washed twice with sterile distilled water and a portion was placed on
a carbon-coated Formvar grid. This was stained with 0.5% phosphotungstic
acid (pH 7-0) and examined in an electron microscope (50KV).
(F) Thin section preparation. Floes were fixed in 1.0% Os04 in veronal
buffer overnight in a refrigerator and stained in uranyl acetate for 2
hours. Dehydration of the floes was performed with a linear gradient
dehydration device (?8) using ethyl alcohol. Each floe was embedded in
Epon 812 monomer according to the method of Luft (6l) and polymerized
by heat. Sections were cut on a Porter-Blum MT-1 Ultra Microtome (Ivan
Sorvall, Inc., Norwalk, Conn.) using glass knives (Ferval Co.) and post
stained with lead citrate.
(G) Bovine serum albumin for phase contrast microscopy. A k2% solution
of bovine serum albumin (Nutritional Biochemicals Corp., Cleveland, Ohio)
was prepared and mixed with cell suspension (1:1) on a glass slide for
the purpose of increasing the refractive index of the suspending fluid.
The wet mount was examined using phase-contrast microscopy (2). Cells
were stained for light microscopy as previously reported
18
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Purification of extrace11ii]ar polymer from culture 115 cells. The cell-
floe matrix was separated from culture supernatant fluid "by centrifuga-
tion at 10,000 x g for 20 minutes and the supernatant fraction was
discarded. The floe pellet was washed once with distilled water, centri-
fuged, and suspended in distilled water (l g wet wt. cells/7 ml water).
The suspension was exposed to ultrasound (Branson Sonifier, 20 KG output
at 3.0 amp. for 5 min.).
The efficiency of matrix removal from cells was determined by examining
the suspension periodically in a phase-contrast microscope using wet
mounts containing cells suspended in 0.03$ crystal violet. The ultra-
sound treated suspension was centrifuged at 8000 x g for 10 minutes to
remove cells and debris. The viscous supernatant fraction was saved,
added to 5 volumes of methyl alcohol and stored at -20°C overnight. A
white flocculent precipitate rose to the surface upon standing. It was
removed with the aid of a watch glass and was air-dried to remove aqueous -
methyl alcohol. The precipitate was dissolved in distilled water at 60°C
with agitation and precipitated in 5 volumes of methyl alcohol. The pre-
cipitate was recrystallized 2 times using the above procedure and air-
dried. The dry material was dissolved in 2 N NH4OH (approximately
100 jig/5 ml), and centrifuged at i»O,000 x g for 3 hours to remove material
insoluble in NH4OH. The water clear supernatant fraction containing dis-
solved polymer was added to an equal volume of 95$ ethyl alcohol. As the
polymer precipitated from the solution it was removed by means of a glass
rod and added directly to a carbon-coated grid for examination in the
electron microscope.
Infrared spectroscopy of purified polymer. The purified polymer was also
added directly to a NaCl crystal under constant exposure to a heat lamp
to facilitate rapid solvent evaporation. The NaCl crystal which was
coated with a thin film of the polymer was examined using a Perkin Elmer
Model 237B Spectrophotometer.
Enzyme susceptibility of purified polymer. Alpha-amylase (Bacillus
subtilus, type IIA, Sigma Chem. Co., St. Louis, Mo.) and beta-amylase
(barley, type IIB, Sigma Chem. Co.) were added separately to purified
polymer and hydrolytic activity was assayed according to the procedures
described by Bernfeld (5).
One ml of an alpha-amylase solution (0.03 mg/ml) was added to 1.0 ml of
polymer solution (0.36 mg/ml of 0.02M NaH2P04 - 0.006M NaCl at pH 6.9)
and replicas were incubated at 20°C for time intervals up to 120 minutes.
One ml of beta-amylase (0.06 mg/ml) was added to 1.0 ml of polymer solu-
tion (0.36 mg/ml of 0.016M sodium acetate, pH U.8) and replicas were
incubated at 20°C for time intervals up to 120 minutes.
One ml of soluble starch (10 mg/ml) was added to both alpha and beta-
amylase solutions in place of polymer to serve as a positive control.
19
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The assays were repeated several times using whole isolate 115 cell-floes
and I-16-M cell-floes in place of purified polymer.
Cellulase (Aspergillus niger, type II, Sigma Chem. Co., 30 mg/15 ml of
0.05M sodium citrate, pH 4.0) was added to whole isolate 115 cell-floes
or I-16-M cell-floes and incubated at 2.k° ± 1°C for periods up to 120
minutes. At desired time intervals, 1.0 ml of Nelson's alkaline copper
reagent was added to each tube and the solution was assayed colorimetri-
cally for sugar as described "by Nelson (70). Carboxymethylcellulose
(12.0 mg/ml) was added to buffered enzyme in place of cells and served
as a positive control.
Ester formation. Cultures of Z. ramigera isolate No. 1 and No. 115 were
maintained on slants of 0.5$ (w/v) Proteose Peptone (Difco, Detroit,
Mich.), 0.5$ (w/v) Yeast Extract (Difco) solidified with 1.5$ agar (Difco)
at 28°C and in Proteose Peptone-Yeast Extract broth at -20°C.
Short chain alcohols, C^ through C8 (including primary and secondary butyl
alcohol), stearyl, and lauryl alcohols were tested in concentrations rang-
ing from 0.1$ to 10$ (v/v) as substrates for ester production and as sole
carbon sources. Culture media ingredients per liter were: (A) Proteose
Peptone (Difco), 0.5 g; Yeast extract (Difco), 0.5 g; ethyl alcohol (95$),
23.0 ml. (B) arginine*HCl, 0.5 g; MgS04-7H20, 2.0 g; 1^HP04, 2.0 g;
KH2P04, 1.0 g; vitamin B^, 1.5 x 10~7 g; ethyl alcohol, 23.0 ml. (c)
(NH4)2S04, 0.5 g; MgSo4-7HOH, 2.0 g; IfeHK)*, 2.0 g; KH2P04, 1.0 g; B^;
1.5 x 10-" g; ethyl alcohol, 23.0 ml.
Ester production in liquid media was investigated in Erlenmeyer flasks,
containing media in volumes equal to 50$ of the flasks' capacities, both
as stationary and shaken (gyratory shaker, 160 rpm, New Brunswick Scien-
tific Co., New Brunswick, N. J.) cultures and in 8 L. of medium in an
aerated fermentor (New Brunswick Scientific Co.). Ester production was
also examined in agar plate cultures incubated at temperatures in the
range of 4°C to ^-5°C in a gradient temperature incubator (Lennox Corpora-
tion, Columbus, Ohio).
Shaken cultures were sampled several times during growth for the produc-
tion of ester and assayed by the two-dimensional thin-layer chromatography
technique of Bleiweiss (6) for intermediary acid compounds. One-dimensional
paper chromatography (Absolute ethyl alcohol:ammonium:hydroxide:water
(160:10:30)) was also employed in the assay of intermediates; acid spots
were located with bromphenol blue spray (O.OU$ w/v alcoholic bromphenol
blue). Microorganic spot tests (Feigle, 37) were employed to detect esters
and intermediate acids. The presence of poly-beta-hydroxybutyric acid,
reported to be present in this organism (Parsons and Dugan, 71) was deter-
mined by light and phase microscopy.
Ester in liquid cultures was detected and identified by a combination of
infrared spectres copy and gas-liquid chromatography (GLC). Five GLC
column packings were tested: 10 ft. Carbowax 600 (10$ w/w) on Chromosorb
¥ (Varian Aerograph, Walnut Hills, Calif.), 10 ft. Carbowax 15^0 (15$ w/w)
20
-------
on Chromosorb W (Varian Aerograph), 3 ft. Hereof lex % on Chromosorb W
(Varian Aerograph), 23 ft. column composed of the latter three columns
in sequence, and 5 ft. ethylene glycol succinate (15$ w/w) on Gas-Chrom P
(Applied Science, State College, Pa.). Nitrogen, with a flow rate of 25
ml/min at the exit port was employed as the carrier gas. Injector and
detector temperatures were l65°C. The column was operated isothermal1y
at ^"C for ester identification. A linear program with a rise of V
to 1^5°C was employed for examination of other culture products. Glass
columns (l.D. 1/8 in) were used in an Aerograph model 20U gas-liquid
chromatograph, equipped with a hydrogen flame detector (Varian Aerograph).
Figure 1 summarizes the procedures followed in concentrating and identify
ing the ester produced by the culture. Cell-free culture supernatant
fluids were directly injected into the GLC column or concentrated before
injection by one of two methods: (A) the cell-free supernate was placed
in a dialysis tube and concentrated by water removal with Aquacide II
(Cal Biochem). (B) the cell-free supernate was continuously extracted
in a counter-current continuous extracter using ethyl ether (anhydrous).
CULTURE
centrifuge 12,000 x g
1
I I
SUPERNATANT CELL PELLET
DISCARD
I I
CONTINUOUS DIALYSIS TUBE
ETHER EXTRACTION
AQUACIDE FOR
EVAPORATE to WATER^REMOVAL
0.1 VOLUME ^X— > EXTRACT DIALYSATE
* DZAL1SATB Wlth f5 MOH
GLC GLC 4 DIRECT
DRY ETOH GLC
with KgCO
I
IR
Figure 1. Flow diagram for ester concentration and identification
21
-------
Cells in mid-log growth stage, grown in the presence and absence of
ethyl alcohol, and their cell-free culture supernatant fluids were
employed in assays to determine the existence of an esterifying enzyme,
either associated with the cells or free in the medium. The proced-
ures are illustrated in Figures 2 and 3.
Culture
Centrifuge
at 12,000 x g
^ Cells - save
for examination (See Fig. 3)
- Supernate —
Add butyric acid
to give 1% cone.
45°C/S min
60°C/S rain
Boil/5 min
Autoclave
Filter sterilize
Add EtOH to each to
give 2% cone. Hold
each of these at:
U-0°, 4°, 20°, 28°,
and 37°C and examine
by GLC for ester
production.
Figure 2. Flow diagram showing procedure used to examine isolate
No. 115 culture media for ester production
22
-------
ro
(jO
Cells
(from Fig. 2)
Wash 2 x in buffer-EtOH,
to remove residual ester
followed by 8 washes in
buffer
Wash in 40C
phosphate buffer
(pH 7.0)
Repeat assays used for
supernate (Fig. 2) with
exception of "filter
sterilize".
Suspend in filter-
sterilized supernate,
disrupt in tissue
homogenizer
Resusnend in buffer,
disrupt in tissue
homogenizer
— Suspend in buffer
Add butyric acid
to 1% cone.
Add butyric acid
to 1% cone.
Suspend in buffer and add 1%
butyric acid
Suspend in sterile
H20
Suspend in filter-
sterilized
supernate (Fig. 2)
Add EtOH to each
to give 2% cone.
- Hold at 28°C and
examine by fiLC
for ester production
Figure 3. Flow diagram showing procedure used to examine isolate No. 115
cells for ester production
-------
Chromatography of polymer hydrolysate. Purified polymer was added to
2N HC1 (1 mg/ml), placed in a glass tube, and sealed. The tube was held
at 100°C for 18 hours. The contents were removed and evaporated to dry-
ness at 9°°C under a stream of air. Two ml of water were added to the
residue and again evaporated to dryness to remove HC1. The evaporation
procedure was repeated three times. The residue was then suspended in
0.3 ml distilled water and the hydrolysate (0.02 ml) was spotted onto
Whatman No. k paper for Chromatography. An ascending two-dimensional
technique was used (phenol-water, U:l, w/v and isopropyl alcohol water
k:l, v/v). Individual chromatograms were sprayed with the following
reagents: aniline-diphenylamine (86), silver nitrate (86), benzidine
(53), aniline-acid-oxylate (23), ninhydrin (86).
The hydrolysis procedure was repeated using 2N H2S04 in place of HC1 and
portions of the hydrolysate were chromatogramed in the same manner.
Gross Adsorption of Metallic Ions
Gross uptake of several colored metal ions by the cells was used as a
screening procedure. Colored metal ions were selected because we could
obtain a visual indication of metal accumulation by the color of the cell
pellet after centrifugation. The following procedure was used: 0.1 ml
of a standardized pre-washed cell suspension in 0.3M phosphate buffer was
inoculated into a series of 250 ml Erlenmeyer flasks, each containing
50 ml of trypticase soy broth (TSB, BBL). The flasks were incubated at
28°C for k8 hours on a reciprocal shaker (100 oscillations/min). The
cells from each flask were centrifuged and washed twice using 10 ml phos-
phate buffer and then resuspended in 50 ml of sterile distilled water in
250 ml flasks. One ml each of stock solutions of CoClg, CuCl^, FeCla,
and NiCl^ were added separately to give the final concentrations of metal
ion per flask shown in Table lU. The cell-metal ion suspension was shaken
at 28°C for 18 hours on a reciprocal shaker and the cells were then sepa-
rated from the solution by centrifugation. Accumulation of colored ions
in the ceH paste at the bottom of the centrifuge tubes was readily seen.
Metal ion concentration in the supernatant was determined via atomic
absorption spectrometry (Perkin-Elmer) and compared to initial concentra-
tions.
Uptake of Zn65
Accumulation of Zn6s by actively growing cells was determined in the
following manner: 0.1 ml of a standardized pre-grown washed cell suspen-
sion was inoculated into 50 ml of TSB in 250 ml Erlenmeyer flasks. ZM65
was added to the flasks in one of three ways: (i) Zn65 to give a final
concentration of l^C in 50 ml growth medium (Sp. Act. 1.11 mC/mg), (ii)
Zn65, l|aC, plus 50 ug carrier Zn as ZnCl^ in 50 ml growth medium, (iii)
Zn65, IfiC, plus 500 ug Zn carrier. The cells were then incubated at
28°C for periods up to lU days on a reciprocal shaker. Flasks containing
the culture were removed from the shaker at specified intervals (see
Figs. 30 and 31) and centrifuged. Five ml aliquots of supernatant were
removed and counted. The cell paste was resuspended in 3 ml distilled
-------
water, mixed by stirring, transferred to a counting vial, and centrifuged
again. The supernatant was decanted and considered as a wash. The cell
paste was dried in an oven at a maximum of l80°C and then weighed and
counted using an automatic gamma well counter (Nuclear Chicago).
Adsorption of Mine Water Ions
Z. ramigera 115 cells were cultivated in 250 ml Erlenmeyer flasks which
contained 50 ml of the following basal growth medium: alanine, 0.5 g;
arginine, 0.25 g; KgHKUs 0.1 g; KH2P04j 0.05 g; MgS04-7H20, 0.01 g;
^2 7.5 x 10-8 g; glucose, 10 g; distilled water to 1 liter. After 72-
hour incubation at 2k°C incubation on a temperature controlled shaker
(180 rev/min), the cell floes were removed by centrifugation and washed
three times with sterile distilled water. The washed cell-floes were
added to 50 ml of prepared ion solutions of known concentration. The
ion-floe suspension was held on the shaker at 2k°C for 6 hours unless
otherwise indicated, after which the floe was removed by centrifugation.
Floes were washed two times with distilled water and the wash water was
saved for analysis. Adsorption of metal ion was calculated by comparison
of supernatant loss, to the concentration in a control flask which con-
tained no cell-floe. Sufficient replicas of each flask were prepared to
give triplicate ion analysis and to allow triplicate determination of
cell-floe wet weight prior to addition of ion solution.
Cations were determined by atomic adsorption spectrometry (Perkin-Elmer)
and anions by standard wet chemical methods.
Adsorption of the Chlorinated Hydrocarbon; Aldrin
Our experimental procedure was as follows: the test organisms were grown
in shake culture at ambient temperature (22° ± 2°C) in nutrient broth
(8 gm/1, Difco), harvested by centrifugation, washed twice and resuspended
in 25 ml of distilled water. Erlenmeyer flasks containing 25 ml suspen-
sions of bacterial floe were then placed on a rotary shaker and 1 ml of
aldrin dissolved in acetone was added to give a final concentration of
1 x 10~6 g aldrin/26 ml. After being shaken at 120 rpm for the time
periods indicated in Figures 37 and 38, the flasks were removed from the
shaker and the floe was separated from the supernatant by centrifugation.
The floes were washed twice with distilled water and the washings were
added to the original supernatant. The pesticide exposure time was cal-
culated as that period between addition of aldrin to the solution and the
separation of the second washing from the bacterial floe. The floe and
supernatant fractions were extracted separately with a 3:1 mixture of
heptane and acetone. The organic solvent fractions containing the aldrin
were concentrated by evaporation at 80°C in glass 15 mm I.D. x 100 mm
tubes and adjusted to a volume of k ml. Two p.1 of sample were injected
into an Aerograph model 200 gas chromatograph (GLC) utilizing an electron
capture detector, 250 me of titanium tri-tritide, column temperature 185°C,
detector 200°C, injector port 225°C, 5 ft glass 1/8 inch internal diameter
column packed with Chromosorb W 60/80 mesh, coated with % Dow Silicone
25
-------
SE-30, high purity N2 gas carrier, 60 ml/min flow rate. The lower limit
of aldrin detection by this procedure was about 2.5 x 10~12 g aldrin as
determined by a 1.0 on peak height on the strip recorder.
Samples of natural sediment which were in the process of settling and
accumulating in Lake Erie were collected by specially designed sediment
collectors placed on reefs (1*9). This sediment consisted primarily of
inorganic clay particles and averaged about 10$ organic matter. In
addition to being analyzed in a manner identical to that described for
bacterial floe, an extract was also examined on a Dohrmann Instrument
Co. model 200A microcoulometer, inlet temperature 300°C, furnace tempera-
ture 800°C, equipped with a 6 ft glass column, 5 mm internal diameter,
packed with Chromasorb W 60/80 mesh, coated with 7.55% of QF-1 and 5.20$
B.C. 200, High purity N2 gas, 190 ml/min flow rate.
Culture and Media for Production of Zoogloea 115 Extracellular Polymer
Zoogloea ramigera 115 as reported by Friedman and Dugan (1*O) and Joyce
and Dugan (56) was employed in this study. The organism was cultured
and maintained in the following modification of the arginine medium of
Crabtree et al. (27): arginine'HC1, 0.5 g; alanine, 1.0 g; MgS04-7HOH,
0.2 g; KgHKU, 2-° S> HE^POif, 1.0 g; carbohydrate, 5-0, 10.0, or 20.0 g;
vitamin B^, 1.5 x 10"6 g; distilled water to 1 liter. Fructose, galac-
tose, glucose, lactose, mannose, soluble starch, and sucrose were
examined individually as the carbohydrate source in the basal medium.
The amount of carbohydrate was varied from 0.5$ to 2.0$ depending on the
experiment.
For each carbohydrate source, polymer was isolated from 100 ml of culture
incubated in 2.0$ carbohydrate in basal medium after 7 to lU days at
2k° ± 2°C on reciprocal shaker (120 strokes/min). To study changes in
batch culture and growth medium parameters, a, lk L. fermentor apparatus
(New Brunswick Scientific, Co. Inc., New Brunswick, N. J.) was inoculated
with a 1$ volume of a 72-hour culture and maintained at 2k° ± 2°C with
continuous agitation (300 rpm) and continuous aeration (3000 cc air/minute)
for periods ranging from 7 to 20 days.
Isolation and Purification of Polymer
Polymer from cells grown on each carbohydrate source was treated sepa-
rately as outlined in Figure k.
Acid Hydrolysis of Dried Extracellular Polymer
One mg of purified polymer and 1 ml of 2W HC1 were sealed in replicate
5" soft glass tubes and hydrolysed at 100°C for either 8.5 or 18 hours.
The hydrolysed material was removed from the sealed tube and transferred
to a 50 ml beaker which was held in a water bath at 90°C while volatile
material was evaporated under a stream of air. A 1.5 ml aliquot of dis-
tilled water was added to the hydrolysed residue and again evaporated.
26
-------
100 ml culture (7 to 14 days)
Add 2 volumes IN NH40H.
Blend 15 seconds.
Centrifuge 1600 x g for
20 minutes.
I
Supernatant(A)
Add 3 volumes cold,
slightly acidified
95% ethanol.
Chill at 0 C overnight.
If polymer is floating,
lift out with a watch
glass. Otherwise,
centrifuge 1300 x g for
60 minutes.
Pellet
Discard.
Supernatant
Discard.
1
Pellet (B)
Redissolve in 1/3 original
volume cold water overnight,
Centrifuge at 1500 x g for
10 minutes
Supernatant
Reprecipitate as in
A above and repeat
steps through B.
(C) Redissolve precipitate
in as small a volume of
water as possible.
Dialyse at 5 C for 72 hours
in double distilled demin-
eralized water changed daily.
Reprecipitate with acidified
ethanol or acetone (2 volumes)
Chill overnight.
Collect polymer and air dry.
Store in desiccator.
Pellet
Discard.
Figure k. Flow diagram outlining procedure for extraction
and isolation of polymer
27
-------
This procedure was repeated three times to completely evaporate the HC1,
then the residue was redissolved in 0.3 ml distilled water and stored
under refrigeration for subsequent paper chromatography.
Paper Chromatography
Descending paper chromatography employing Whatman No. h paper strips,
19 by 80 cm, and a cylindrical glass tank equilibrated at 2h° ± 2°C
was used to identify polymer components in the hydrolysed samples. Known
samples (100 |j,g/ml) and polymer hydrolysates (0.1 ml) were spotted on
each sheet. Three solvent systems were selected for use: n-butanol-
acetic acid-water (^:1:5 v/v), iso-propanol-water (^-:1 v/v), ethyl
acetate-pyridine-water (12:^:5 v/v). Location reagents included naph-
thoresorcinol dip (0.2% in acetone, 1 volume to 1 volume of 9$ phos-
phoric acid in water), silver nitrate spray (0.3M AgNo3 in 5M NH4OH),
benzidine dip (0.1 g in UO ml glacial acetic acid, 30 g trichloroacetic
acid, and UO ml water, 1 volume to 9 volumes acetone), and ninhydrin
spray (1.0 g ninhydrin in 500 ml n-butanol, before use add 0.5 ml
collidine).
Fermentor Studies and Viscosity Peterm-ination
At each sampling time approximately 150 ml of whole culture were removed
from the fermentor. Two 10 ml aliquots of whole culture material were
quick frozen at -70°C for subsequent determination of dry weight, total
deoxyribonucleic acid (30), and po3y-beta-hydroxybutyric acid (58).
Two 10 ml aliquots of supernatant (6600 x g for 10 min., Sorvall SS-l)
were frozen at -70°C for subsequent determination of Nelson's reducing
sugar (2k) and anthrone sugar (88). An 8 ml aliquot of whole culture
and an 8 ml aliquot of the supernatant were used for viscosity determi-
nation to estimate polymer production. Precalibrated (#50 B801, #200
Y823, #500 392) Cannon-Fenske Routine Viscometer tubes (Cannon Instrument
Co.) were held plumb in a water bath at 23° ± 3°C. Duplicate time deter-
minations were recorded for each sample. The average time in seconds for
each sample was multiplied by the temperature corrected viscosity con-
stant for each tube to obtain viscosity in centistokes.
28
-------
SECTION V
13MENTAL RESULTS AND DISCUSSION
Nutrition, Biochemistry and Morphology
Several floe-forming bacteria were isolated in this laboratory and others
were obtained at various times during the five year span of this investi-
gation from investigators at other laboratories. These isolates were
examined using standard microbiological techniques for identification and
characterization. Because a.n isolates were not available during the
initial phases of this investigation some have been more thoroughly
studied than others and in some instances the organisms were not examined
in this laboratory with regard to biochemical characteristics.
Table 1 lists the floe-forming isolates examined during the course of
this study and Tables 2, 3 and k list results of biochemical reactions
for specified floe-formers. During the course of this investigation
attention was focused on two closely related isolates of Zoogloea ramigera
i.e. isolate 115 and isolate 1. Further biochemical data comparing these
isolates are presented in Tables 5» 6, 7 and 8.
Crystal violet decolorization is listed as a differential criterion for
classification because it was observed that some of the isolates behaved
differently when grown on crystal violet agar. Strain 115 is the only
isolate observed to decolorize crystal violet; C-l, C-3 and C-22-U fail
to develop colonies on the medium; and I-16-M, P-8-U and P-95-5 adsorb
the dye to produce violet colonies.
The color indicator bromothymol blue is a very sensitive acid indicator
and it should be stressed that values recorded in Tables 2, 3 and k as
VSA are not significantly acid but merely represent a change from the
initial pH (7.2) of the medium toward acidity. The difference between
values recorded as + and VSA are therefore probably not highly signifi-
cant.
Comparison of organisms. All isolates are gram negative, polar flagel-
lated rods which flocculate and contain sudanophilic granules under
certain growth conditions. None of the Crabtree isolates (I-16-M, P-S-^,
P-95-5> C-22-U) have been shown to produce a zoogleal matrix or capsules.
Strain 115 of our isolates has been shown to possess a zoogleal matrix,
whereas, C-l and C-3 have not.
Strain I-l£-M has been recommended by Crabtree and McCoy as a neotype
Zoogloea ramigera and comparison of this strain to P-8-U, P-95-5, and
C-22-4 has been reported previously (27, 28). C-3 differs markedly from
all other isolates with regard to biochemical reactions and is considered
to be a flocculent Pseudomonas species.
29
-------
TABLE 1. Floe-forming isolates, source of isolation and
reference to information if available
Isolate
Zoogloea ramigera 1
Z. ramigera 115
Z. ramigera I-16-M
Z. ramigera Z-SC-38
Z. filipendula P-8-4
Pseudomonas denitrificans P-95-5
P. denitrificans ATCC 13867
Acetobacter suboxydans ATCC 621
A. oxydans ATCC 6433
Unidentified gram negative
polarly flagellated
C-l
C-3
C-22-4
Obtained from
Dug an
Dug an
Crabtree-McCoy
Ganapati
Crabtree-McCoy
Crabtree-McCoy
ATCC1
ATCC
ATCC
Dugan-Friedman
Dugan-Friedman
Crabtree-McCoy
Reference
56
40, 42, 43
27, 28
44
26, 27
26, 27
ATCC
ATCC
ATCC
40
40
26, 27
*ATCC, American type culture collection
30
-------
TABLE 2. Biochemical reactions of floe-forming bacteria6
Organism
Zoogloea
ramigera
115
C-l
C-3
£• rarcigera
I-16-M
Z. filipendula
P-8-4
Pseudomonas
den itrif lean:
P-95-5
C-22-4
Litmus milk
Slightly alkaline
Alkaline
Acid
Slightly alkaline
Slightly alkaline
Slightly alkaline
Slightly alkaline
Methyl
red
-
-
*
-
„
-
Nu-
trient
gelatin
-
-
*
-
_
m
-
Ureas e
*
-
-
-
+
-
Indole
-
-
4-
-
^
-
Cit-
rate
-
*
-
-
—
-
N03
re-
ducer
*
+
-
-
+
+
*
N03
N2
-
-
-
-
^
-
Casein
hy-
drolysis
*
-
*
-
—
-
Starch
hy-
drolysis
*
-
*
-
-p
-
Crystal
violet
decolor-
ized
*
NG
NG
-
—
NO
Cat-
alase
*
*
+b
*
*
.
*
U)
H
aAll organisms were cytochrome oxidase positive. None produced sulfide. Reactions are indicated as follows:
-, negative response; *, positive response; NG, no growth.
slight.
-------
TABLE 3- Reaction of floe-forming bacteria in sugar broth with bromothymol blue indicator
Organism
Zoogloca
ranigeca
115
C-l
C-3<
I-16-M
Z.. filipendula
P-8-4
Faeudomonas
denitrtflcana
P-9S-S
C-22-4
Arabi-
noso
VSA«
VSA
A8
A
*
SA
A
Cello-
>iose
VSA
VSA
A
»
+
*
*
Fruc-
tose
VSA
VSA
A
VSA
^
SA
A
Galac-
tose
SA"
»
A
VSA
+
SA
A
Glu-
cose
+c
VSA
A
VSA
#
*
A
Glyco-
gen
SA
*
A
4,
+
*
Inu-
lln
*•
VSA
*
+
4
*
Lac-
tose
VSA
VSA
A
*
*
Levu-
lose
VSA-
*
A
VSA
+
SA
SA
Mal-
tose
SA
VSA
A
SA
.
SA
VSA
Man-
nose
VSA
VSA
A
«.
*
VSA
SA
Melezi-
tose
*
VSA
*
.
*
*
Meli-
biose
*
VSA
*
,
.
*
•
Raffi-
nose
*
VSA
*
»
+
*
Rham-
nose
4
VSA
*
«.
.
SA
Sali-
cin
»
VSA
A
4
.
*
*
Sor-
bose
*
VSA
*
*
^
4
Suc-
crose
VSA
VSA
A
SA
VSA
SA
Xylose
*
VSA
*
VSA
4
SA
SA
LO
ro
•Very slightly acid, blue green, pH 6.9 to 6.8.
"Slight acid, yellow green, pH 6.7 to 6.3.
ci
-------
TABLE k. Reaction of floe-forming "bacteria in alcohols and sugar alcohols
using bromothymol blue indicator
Organism
Zoogloea
ramigera
115
C-l
C-3
Z. ramigera
I-16-M
Z. filipendula
P-8-4
Pseudomonas
denitrificans
P-95-S
C -22-4
Methanol
1%
•f
+
+
+
+
+•
+
Ethanol
1%
+
VSA
+
+
VSA
+
A
Propanol
1%
•f
VSA
*
+
VSA
•f
VSA
Butanol
1%
+
+
•••
+
+
+
+
Glycerol
1%
+
VSA
A
4-
•f
VSA
+
Inositol
+
VSA
+
+
+
SA
+
Mannitol
•«•
VSA
A
VSA
•f
VSA
SA
Adonitol
+
VSA
+
•f
•f
SA
+
Sorbitol
+
VSA
+
+
•f
SA
SA
Dulcitol
+
VSA
+
+
*
*
•»•
to
U)
+, growth but no acid, blue
SA, slight acid, yellow green
+, slight growth but no acid, blue
A, acid, yellow
VSA, very slight acid, blue green
-------
TABLE 5. Biochemical characteristics of Zoogloea isolate No. 1 and
Zoogloea ramigera isolate No. 115 when cultured in liquid
tubes of purple broth basal medium (Difco) or semi-solid
media at 25°C
Test
Isolate No. 1
Isolate No. 115
Litmus milk
MR, VP
Gelatine hydrolysis
Gelatin liquefaction
Collagen hydrolysis
Chitin hydrolysis
Urease
SIM (sulfide, indole, motility)
Citrate, Koser's broth
Citrate, Simmon's slant
Nitrate (N03 to N02)
Starch hydrolysis
Casein hydrolysis
Catalase
Crystal violet
Alka
+b in 72 hr
+ in 4 weeks
Growth in 24 hr
Alk, 24 hr
Decolorized
Alk
+ in 72 hr
+ in 4 weeks
Weak + after S
days
Growth in 24 hr
Alk, 24 hr
Decolorized
aAlk » alkaline (color change purple to deep blue except in the case of Simmon's
citrate slant which went from green to deep blue).
a positive reaction or presence, (-) « negative reaction
-------
TABLE 6. Growth of isolates No. 1 and No. 115 in basal medium II
plus amino acids as carbon and nitrogen sources; cultures
were incubated at 25° C for 72 hr
Substrate
Added
Alanine
Arginine
Aspartate
Cystine
Glutamate
Glycine
Histidine
Homoserine
Hydroxyproline
Isoleucine
Leucine
Lysine
Methioninc
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyros ine
Valine
Guanine
Adenine
Cytosine
Basal Medium II plus
Supplement B.-a
Isolate No. 1 115
-d 4-
4
4-
4- 4-
_
•f
4-
•f
I If
_ .
_
•f
•f
4-
*
YEb
1 115
- I
~ ~
_
*e
4-
- *
_ _
.
I +
+
4.
•f
YE + Glucose0
1 115
• *
•f 4-
_ _
*
4-
- *
. .
_ _
4-
+
4.
*
aBj2 concentration: 1.5 x 10"' pg/ml.
''Yeast Extract concentration: 10 ug/ml.
°YE concentration: 10 |ig/ml; glucose concentration: 20 mg/ml.
^(*) - growth, (-) = no growth.
eWhen this medium was supplemented with ethyl alcohol, formaldehyde was produced.
*Very slight growth after 72 hr.
35
-------
TABLE 7. Growth of isolate No. 115 with sugars and sugar
alcohols as sole and supplementary carbon sources;
cultures were grown at 25° C for 72 hr
Substrate
Added*
Arabinose
Cellobiose
Cellulose
Fructose
Galactose
Glucose
Lactose
Maltose
Mannose
Melibiose
Melezitose
Raffinose
Rhamnose
Ribose
Sorbose
Sucrose
Xylose
Arabitol
Dulcitol
Erythritol
Glycerol
Inositol
Mannitol
Sorbitol
Basal Medium II plus
(NH4)2S04b
+d
+
-
+
+
+
+
+
+
+
+
+
+
+
-
+
+
•f
+
+
+
+
•»•
+
Argininec
+
+
-
+
•»•
•f
+
+
•*•
+
+
+
•»•
+
+
•f
+
•f
+
+
+
+
+
•f
aSugar and sugar alcohol concentrations: 50.0 mg/ml.
° concentration: 0.5 mg/ml.
Arginine concentration: 0.5 mg/ml.
(+) a growth, (-) * lack of growth.
36
-------
TABLE 8. Growth of isolate No. 115 on short chain alcohols
and acids -when added to basal media
Substrate
Addeda
Methyl alcohol
Ethyl alcohol
Propyl alcohol
Isobutyl alcohol
sec-butyl alcohol
n-Amyl alcohol
Dodecyl alcohol6
Lauryl alcohol6
Stearyl alcohol
Ethylene glycol
Diethylene glycol
Formic acid
Acetate (Na)
Propionate (Na)
Butyric acid
Ethyl butyrate
a-ketobutyric acid
a-ketoglutaric acid
P-hydroxybutyric acid
Caproic acid
Capri c acid
Laurie acid
Stearic acid
Basal Medium II plus
(NH4)2S04b
+d
4
4
4
-
4
_
-
-
4
-
_
4
4
4
-
-
-
4
.
.
.
•
Timec
<24
<24
<24
<24
72
48
24
48
120
120
Arginine
*
4
*
4
-
4
+
-
-
4
-
4
4
+
4
4
•f
4
4
4
4
.
—
Timec
< 24
< 24
< 24
< 24
36
40
48
< 24
24
48
120
240
168
168
120
3!
3f
aAlcohols and acids were tested in 0.5%, 1.0%, and 2.0% concentrations and
^showed no variation in results on any concentration.
concentration: 0.5 mg/ml; arginine concentration: 0.5 mg/ml.
^Approximate time in hours of appearance of growth.
(*) * growth, (-) * no growth.
6Two different suppliers: dodecyl alcohol from Aldrich Chemical Company, Inc.;
lauryl alcohol from Merck and Company.
Time in weeks.
37
-------
C-l, I-16-M, and 115 all differ in at least two biochemical character-
istics. Of these three strains only I-16-M produced a significant acid
reaction on any of the carbon sources examined. I-16-M appears to
ferment arabinose, galactose and maltose to a lesser extent.
Isolate 115 which forms a typical gelatinous or zoogleal matrix also
differed from I-16-M in ability to degrade urea, starch, casein, and
crystal violet dye. Colonial morphology of 115 is quite similar after
2k hours incubation to colonies of I-16-M. However, 115 colonies develop
a rugose appearance upon further incubation (kO-72 hr), whereas the
colonial appearance of I-16-M remains the same. The photographs shown
in Fig. 5 are typical of the colonial morphology of the two isolates
being compared. A depressed center is characteristic after 18-2^ hr
incubation but becomes wrinkled with a moist appearance beyond 2k hr
incubation.
Figure 6 illustrates the capsule which surrounds Z. ramigera 115 cells
which results in the formation of zoogloeal slime and "finger-like"
projections often described as being characteristic of the organism.
Figures 6a and 6b show the typical "finger-like" projections often
observed in the slime from a trickling filter or other quiescent natural
water that contains organic contaminants. Figure 6c is the same organism
under phase contrast microscopy after being negatively stained by the
Maneval technique. The cells are grouped in individual packets of two
or four cells surrounded by capsules and the individual clumps adhere
together forming a "finger-like" projection. The same phenomenon can
also be seen in Fig. 6d which is prepared in the same manner as 6c but
photographed under brightfield illumination. Figure 6e is similar to
6d and shows greater build-up of "finger-like" projections. A typical
zoogloeal mass of capsular slime and embedded cells can be seen in
Figs. 6f, 6g and 6h. Granules of poly-beta-hydroxybutyric acid are
located within the cells which cause them to appear pleomorphic (Figs.
6e, 6f, 6g and 6h).
Z_._ rarrn'gera I-16-M is shown in Fig. 7a. No extracellular material is
observable around I-16-M when grown on the same medium as 115 (Fig. 6),
but intracellular granules previously reported to be IHB are present (27).
Morphological characteristics of isolate C-3 are shown in Fig. 7b. No
extracellular material has been observed, but large quantities of intra-
cellular material accumulate. Continued synthesis of intracellular
granular material causes pleomorphism. Since the granule synthesis is
related to nutrient and growth conditions, cell size and shape are not
desirable characteristics for identification purposes.
Utilization of defined media. Further comparison among I-16-M, 115 and
C-3 were made using the defined media A, B and C listed in the section
describing characterization of organisms. All of the defined medium
variations examined supported flocculent growth of the three isolates
although flocculation was not quantitatively consistent. For example,
115 and I-16-M gave the greatest amount of flocculent growth when the
38
-------
Figure 5. Colonies of Zoogloea ramigera 115 and I-16-M after
cultivation on tryptone glucose extract agar at 28°C.
All colonies are straw colored and appear to be viscous,
but are tough and leathery. (l) 115 after 2k hr show-
ing glistening appearance. X5. (2) 115 after 30 hr
showing characteristic depressed center of "donut"-shaped
colony. X5. (3) 115 after ^0 hr showing the rugose
appearance. X5. (*0 I-16-M after 72 hr showing the de-
pressed center and a crenated periphery. I-16-M does
not develop a completely rugose appearance. XT
39
-------
t''V~:^?*'
• v?ii-JVM **;
Figure 6. Photomicrographs of isolate No. 115 oells show-
ing "finger-like" projections ia,b); individual cell
packets that make up the finger-like projections
under phase contrast illumination (c), and under
bright field illumination (d,e). 6f, g and h show
zoogloeal masses often referred to as the zoogloeal
matrix
-------
• •- Wk» _++
,-v 1^
Figure 7. Photomicrographs of a floe of Z. ramigera I-L6-M cells
(7a) and C-3 cells (?b) showing lack of observable
zoogloeal matrix or capsule around cells
B
-------
media were supplemented with glucose and vitamins. C-3 produced more
flocculent growth in the arginine medium (A) when no supplement was
added, but it did flocculate in the supplemented medium. The casamino
acid medium (c) allowed more rapid growth of the bacteria. The simplest
medium which promoted the most flocculent growth was (A) supplemented
with glucose and B12. This medium was used in most subsequent bio-
chemical studies. It should also be noted that the cultures grew well
in the presence of ammonium ions (medium B) which is contrary to the
data of Rich (82).
The generic name Zoogloea is derived from Greek roots and literally
translates as "living glue". It seems apparent that descriptions in
the literature are based upon microscopic observations of the gelatin-
ous matrix or zoogleal material and that this was the unique character-
istic of the organisms upon which classification resided. Cultures
which do not possess a gelatinous matrix may flocculate but would not
necessarily be Zoogloea bacteria; that is, floe formation and zoogleal
growths are not synonymous. We prefer to consider zoogleal formation
as highly polymerized exocellular material analogous to a capsule, and
floe eolation as a clumping of cells probably resulting from cell sur-
face attractions which probably involve physiochemical influences of
capsular material or extracellular polymer fibrils.
Cell size and shape, as well as gelatinous material production, varies
considerably with culture media, incubation temperatures, and perhaps
oxygen tension in liquid culture media. However, it should be stated
that oxygen tension varies with temperature and one influence is not ex-
clusive of the other in our test system. Cell size and shape within
limits are, therefore, not adequate criteria for characterizing Zoogloea
species. Comparison of the Crabtree isolates to our isolates, using
identical procedures, shows that a similar pattern of carbohydrate re-
actions exist among cultures which do produce a gelatinous matrix (115)
and those which do not (I-16-M). It is also apparent that cells which
flocculate and appear similar morphologically under most growth condi-
tions, do not have similar biochemical reaction patterns (C-3 and 115).
That is, C-3 appears to be a Pseudomonas or Aeromonas species which
flocculates. Strain 115 by virtue of the presence of a zoogleal matrix
is a Zoogloea species according to recognized systems of classification
(10). By comparison of 115 to I-16-M, one must conclude that I-16-M is
either a nonzoogleal producing Zoogloea, as reported by Crabtree and
McCoy (29) or a Pseudomonas species.
The biochemical reaction patterns of 115 and I-16-M appear to be suffi-
ciently different to conclude that they are different species. However,
Unz has isolated 1^7 strains of floe-forming bacteria based primarily on
biochemical differences, all of which he considers to be Zoogloea
species. (Ph.D. thesis, Rutgers Univ., New Brunswick, New Jersey).
One further point we wish to make regards the observation that both 115
and I-16-M deposit high concentrations of iron around the floes if iron
salts are added to the growth media. Since 115 has a gelatinous matrix
around which iron is deposited, one must consider the possibility that
U2
-------
it is a species of either the family Siderocapsaceae or Caulobacteraceae.
A discussion of the relationships among iron depositing bacteria and the
gelatinous matrix of zoogloea producers is therefore warranted.
DeToni and Trevisan (29) used a gelatinous matrix as a criterion for
differentiating sub-families of Coccaceae. A subsequent classification
of the same groups of "bacteria by Winslow and Rogers (100) used the
phrase: "cells aggregated in groups, packets or zoogloea masses" for
differentiating all known cocci. Buchanan (9) later suggested that the
genus Siderocapsa might be sufficiently distinct from other cocci and
presented a new key which included Siderocapseae. In 1929 Pribram (80)
reported on bacterial classification and recognized both rod and coccoid
forms of Siderocapsaceae. He used the term "zoogloea" although he did
not recognize the genus Zoogloea. Drake (31) has recently presented a
discussion of Siderocapsa treubii and considers the work of Hardman and
Henrici (k6) pertaining to occurrence, distribution, and growth of
Siderocapsa in alkaline iron bearing waters.
With reference to a possible relationship between Zoogloea and species
in the order Caulobacteriales a Henrici and Johnson (48) described the
stalked iron depositing bacterium Nevskia on the basis of lobose stalks
composed of gum forming zoogloea-like colonies. These latter authors
published photomicrographs which show a remarkable similarity to those
shown of 115 in Fig- 6.
It is evident that zoogloea producing bacteria are common and ubiquitous
in aquatic habitats. Classification of Zoogloea is presently based upon
highly artificial criteria such as presence of a gelatinous matrix, which
is dependent upon growth conditions. Classification of Siderocapsaceae
is also dependent upon artificial criterion such as deposition of iron
around the gelatinous matrix in the natural habitat. This' is dependent
upon iron content of the natural water environment. We would like to
suggest the possibility that investigators in the past would consider
the same bacterium as a Siderocapsa if it was observed in iron bearing
water, or as Zoogloea if it was isolated from water rich in organics but
extremely low in iron content. Isolate 115 will deposit copper, cobalt,
nickle, and presumably other ions in the gelatinous matrix (see subse-
quent section of report). If these cations had been present in suffi-
cient concentrations in the environment we would have considered it in
a different manner.
It is possible to interpret Fig. 6 as a collection of generally spheri-
cal-shaped clumps of bacteria, each surrounded by a gelatinous matrix
similar to the growth pattern now recognized for Siderocapsa species.
Adherance of these spherical gelatinous masses would explain the make-
up of what has historically been referred to in Zoogloea literature as
finger-like projections.
Table 5 presents comparative data obtained from the two isolates using
standard biochemical tests in addition to hydrolytic reactions on
naturally occurring polymers.
1*3
-------
Both isolates were capable of using ammonium salts as the sole source of
nitrogen when glucose or ethyl alcohol was supplied as a carbon source.
The ability to use amino acids varied in the two isolates. As can be
seen in Table 6, isolate 1 was nutritionally more demanding than No. 115.
It is interesting to note that growth of isolate 115 on hydroxyproline
used as the sole carbon and nitrogen source possessed no odor, but pro-
duced an aldehyde odor when supplemented with ethyl alcohol. This product
was identified as an aldehyde by the Schiff test (21) and as formaldehyde
by the chromotropic acid test (22). No acetaldehyde was detected by the
iodoform test (21).
Isolate 115 was capable of utilizing many sugars and sugar alcohols as
anticipated from the many published descriptions of Zoogloea (27, 1*Q, 93,
9^). Table 7 shows the results of tests made to determine the ability
of this isolate to use the sugars and sugar alcohols as sole or supple-
mentary carbon sources. Of the compounds examined, only cellulose and
sorbose were unable to serve as sole carbon source. Sorbose did not
inhibit growth in the presence of arginine. Presence of cellulose pre-
vented growth in presence of arginine under the experimental conditions.
We assume this was due to adsorption of low concentrations of nitrogen
or cations by cellulose and not direct inhibition.
Table 8 illustrates the ability of isolate 115 to grow on certain short
chain alcohols and acids. The organism eventually grew on most of these
substrates tested, but the incubation times required before there was
visual evidence of growth varied considerably, and probably reflects an
induction period for certain substrate utilizations. When lauric and
stearic acids were incorporated in media which normally allowed abundant
rapid growth (e.g., PFYE), no growth could be detected at the end of a
Ij-week incubation period. Ethyl alcohol allowed rapid appearance of
growth. The presence of short chain alcohols did not significantly alter
the time required for growth to appear nor the quantity of growth as
compared to growth in media devoid of alcohols. The presence of short
chain acids (propionic, butyric, the alpha-keto acids, and the ester
ethyl butyrate), however, resulted in a lag period of 2 to 7 days before
growth appeared. Caproic and capric acids inhibited growth in the ammonium
salts medium but allowed slow growth (3 weeks) in the arginine medium.
Lauric and stearic acids appeared to inhibit growth completely in both
media.
Figure 8 illustrates the enhancement of growth in ethyl alcohol supple-
mented medium over that in nonsupplemented medium.
This suggests that ethyl alcohol is contributing to cell-floe mass
(Klett Units) in proportion to alcohol concentration and is independent
of the other substrates (PPYE), which is suggestive of growth control
by substrates other than.carbon.
Because of our previous observation of rapid metal adsorption by cell-
floes (Ul), we decided to examine the adsorption of organic compounds by
floes. The amino acids alanine and arginine were selected for study.
-------
0.10
8
0_
~_ 0.08
tr w
h; o o,06
-Jo.04
0.02
PPYE
PPYE + 2% EtOH
3 5 7 9 II 13 15 17 19 21
KLETT UNITS
Figure 8. Relative growth (KLett Units) of isolate 115 versus
substrate (PPYE) concentration in the presence and
absence of 2$ ethyl alcohol after ^8 hr at 25°C and
pH 7.0
74 ARC.
6.7 ALA.
4 8 12
Time(hrs.)
Figure 9« Concentration of arginine or alanine remaining in
solution, after removal of cell-floe, versus time.
pH of each solution is shown at each time interval
-------
Replicate samples, each containing approximately 20 gm of washed resting
cell-floe gel (approx. 0.1 gm dry wt. ), were suspended in 50 ml of amino
acid solution in Erlenmeyer flasks and held at 2h°C on a rotary shaker.
Flasks were removed after U, 8, and 12 hours and the concentration of
amino acid was determined in the supernatant after removal of the floc-
gel by centrifugation.
Figure 9 shows the concentration of amino acid (quantitative ninhydrin
colorimetric reaction) versus time. Approximately ^0% of the available
amino acid was removed from solution within k hours. The concentration
remained static between k- and 8 hours and decreased further between
8-12 hours. The pH of supernatants showed no significant change between
0-k hours.
We interpret the loss of amino acid between 0-k hours as adsorption with-
out significant metabolic activity on the amino acid. A rise in pH is
observed between k and 8 hours, which we interpret as HH3 liberation due
to amino acid oxidase activity on the adsorbed amino acid. After 8 hours,
sufficient metabolic activity would be present because ami no acid degra-
dation would supply nutrient to the cells. Between 8 and 12 hours the
pH increases further in correspondence with further decrease in amino
acid concentration. We are aware that these projections are not conclu-
sive since data on cell respiration are not yet available.
Zoogloea rami gera isolates 1 and 115 are typical of the floe-producing
organisms encountered in aerobic waste treatment processes. Isolate 115
is capable of metabolizing a variety of amino acids as both carbon and
nitrogen sources, whereas 1 could not use amino acids, except histidine,
as nitrogen sources. Ami.no acid utilization is accompanied by a pH rise
in the medium, presumably due to NHt liberation. Both isolates were able
to hydrolyze casein, gelatin, starch and urea, and isolate 115 could
hydrolyze collagen.
A more detailed examination of isolate 115 indicated that the organism
could use ammonium as a sole nitrogen source and a variety of organic
acids, alcohols, sugars, and sugar alcohols as sole carbon sources. • This
suggests that the organism probably converts proteinaceous waste material
by hydrolysis and amino acid oxidases to corresponding acids or keto acids
plus EHt. The acids would be further metabolized using NHt as the nitro-
gen source. The organisms are also capable of nitrate reduction, which
could produce a suitable nitrogen source in the presence of suitable
carbon substrates.
C12 and C18 alcohols and acids were inhibitory to isolate 115 in the con-
centrations tested (0.5$ = approx. 0.03 molar). Although these concen-
trations are not likely to be encountered in waste treatment systems,
some inhibition might be expected from high fat content waste water.
These, and related bacteria, have been shown to synthesize and store
large quantities of poly-beta-hydroxybutric acid (27, 73) and to syn-
thesize extracellular polysaccharide matrix fibrils from a variety of
-------
carbon sources (Uo, Ul). This synthetic activity represents a BOD sink
or removal mechanism when the flocculent growth settles from suspension.
Isolate 115 has also been shown to produce butyric acid from ethyl
alcohol. It is then ester if ied with residual ethyl alcohol in the
growth medium to produce ethyl butyrate. Other esters may be produced
by the organisms, depending upon substrate availability, although this
has not been studied. Ester if ied acids would not be recognized as net
acid accumulation in growth media or waste water and could be "stripped"
from the system by aeration mechanisms. These bacteria in a mixed
natural population could be regarded as "buffer" bacteria in that they
would neutralize acids produced during high carbon to nitrogen growth
conditions (see next section).
The exocellular polymers synthesized by the floe formers have a high
capacity for adsorption of metal ions (Ul) and organic compounds such
as ami no acids. The adsorption of organic compounds to matrix polymer
is analogous to the biosorption described in activated sludge processes
by Eckenfelder (see 6k] and can explain the exceptionally high rate of
BOD removal reported by Butterfield (lU, 15) for these organisms.
Ester Synthesis from Alcohol
Growth on alcohol supplemented media. Growth was obtained within 2k
hours at 28° C on all media supplemented with C 1 through C 5 alcohols
at concentrations below 5$. Higher concentrations appeared inhibitory.
Alcohols of length C 6 through C 8 also permitted growth, but the
growth was considerably slower, requiring as long as one week to reach
the levels obtained with shorter chain alcohols. Lauryl and stearyl
alcohol appeared to inhibit growth completely. Short chain alcohols
(C 1 through C 5) also could serve as the sole source of carbon in the
synthetic medium containing ammonium salts and vitamin B^ -
Ester identification. During growth on alcohol supplemented media only,
a fruity odor, typical of esters was detected. In the case of ethyl
alcohol supplemented media the odor was described as that of fresh pine-
apple. An infrared spectrum of an absolute alcohol extract of an
Aquacide culture supernate concentrate is shown in Fig. 10. Fig. 10B
represents the spectrum of an authentic ester (ethyl butyrate, 1$ v/v).
It can be seen that both spectra show doublet absorption bands at 1720
and 1730 cm-1 and a band at 1180 cm-1. These bands are in the regions
assigned to the carbonyls of ester groups.
Table 9 lists the retention times of authentic esters and the unknown
ester produced in the Z. ramigera culture system on an ethylene glycol
succinate column at 75TC. It can be seen that ethyl butyrate was the
only authentic ester possessing a retention time identical to the
culture ester. Similar results were obtained with other columns tested
and at lower temperatures.
-------
CD
25
100
80
uj60
^40
2: 20
MICRONS
3.0 3.5 4.0 5.0
T—1—I I I I | I I—I | I I ULU*»f
MICRONS
6.0 7.0 8.0 IQO 11.012.0 16.0
"i—| i i i i i I i i i 11II i Mi i i I i 11 in
UNKNOWN
_L
4000 3500 3000 2500
2000 1800 1600
XcirT1
(A)
1400 1200 1000 800
MICRONS
3.5 4.0 5.0
MICRONS
6.0 7.0 8.0 10.0 II.OIZO 16.0
I I I I I I I I I I I I I I I I I I I I I I I I I I I | I I I I I I I
4000 3500
3000 2500 2000 1800 1600 1400 1200 1000 800
cm'1
(B)
Figure 10. Infrared spectra of A) alcohol extract of Aquacide concentrated
culture supernatant; B) control ethyl butyrate. Doublet bands at
1720 and 1730 cm"1 and a band at Il80 cm"1 (asterisks) are in the
region assigned to the carbonyls of ester groups
-------
TABLE 9« Values showing GLC retention times of selected
esters on a 15 percent EGS column at 75°C
Ester, 1% Time (sec)
Methyl propionate 98
Methyl "butyrate 98
Ethyl propionate 105
Ethyl butyrate 126
Ethyl propionate 210
A13yl caproate 371
Capryl acetate 315
Culture ester 126
TABLE 10. Values indicating the shortest length of time
required for ester detection in culture systems
compared to acid catalyzed ester formation in
the absence of culture; solutions were held
at 25°C.
System plus 2.% ethyl alcohol Time (hr)
Propionic acid + cone. H2S04
Butyric acid + cone. H2S04
Caproic acid + cone. H2S04
Culture (pH 6.8)
-------
The response to direct injection of cell-free culture supernates from
a PPYE and PPYE-alcohol grown cells into an EGS column at U5°C is shown
in Fig. 11. It can be seen that the only response to the supernatant
from the non-supplemented media (A) occurred after 360 sec. as a broad
peak or series of peaks. The alcohol supplemented culture (B) yielded
peaks with maxima at 10^ and llj-9 sec. and a broad response from ^83 to
720 sec. The response at 10U sec. corresponds to ethyl alcohol (identi-
cal retention time with authentic sample) while the response at 1^9 sec.
represents the culture ester. The responses occurring after 360 sec. in
both curves may be due to fatty acids (as suggested by the response to
injection of a mixture of unmethylated fatty acids - formic, acetic,
propionic, butyric, and lactic - in distilled water) or to as yet uniden-
tified esters present in low concentrations. Figure I2A illustrates the
response to injection of an Aquacide concentrate of alcohol supplemented
culture supernate. Peaks corresponding to ethyl alcohol and the ester
are seen. Addition of authentic ethyl butyrate (0.5% v/v) reinforced
the ester peak as seen in Fig. 12B while addition of 2% (v/v) ethyl
alcohol reinforced the alcohol peak (Fig. 12C). Figure 13A shows a simi-
lar response to injection of an ester extract of alcohol supplemented
culture supernate. The alcohol and ester peaks are visible. Figure 13B
illustrates the reinforcement of the ester peak when the extract was
co-chromatographed with authentic ethyl butyrate (10$).
Nature of ester production. A comparison of the times required for
production of ethyl butyrate, in quantities detectable with GLC, by
spontaneous esterif ication of ethyl alcohol and butyric acid in the
presence and absence of suifuric acid and in the presence of non-alcohol
and alcohol grown cells of Z. ramigera 115 is shown in Table 10. The
data indicate the formation of ester in the presence of alcohol grown
cells occurs more rapidly than in the absence of cells (spontaneous
esterif ication) or in the presence of non-alcohol grown cells. Reddy
(1969) reported the formation of ethyl butyrate by Pseudomonas fragi
in milk to be quantitatively enhanced by the inclusion of ethyl alcohol
and butyric acid in the system. In the case of Z. ramigera, inclusion
of butyric acid did not appear to speed or quantitatively increase the
production of the ester.
The pH of the culture system was in the range of 6.7 to 8.5 when the
ester was detected by GLC while the chemical process occurred at pH
levels below 6.5. Figure lU illustrates the pH changes occurring during
growth of isolate 115 in media, both alcohol supplemented and non-
supplemented. The pH of the non-supplemented and supplemented media
dropped from 7.0 to approximately 6.5 during the first four hours. The
non-supplemented cultures remained slightly acid for hO hours when the
pH suddenly rose, probably reflecting the formation of ammonium ions due
to protein or amino acid degradation. The pH of the alcohol supplemented
media rose quickly after the initial drop, reaching a maximum of 8.5
after ^Q hours. This pH rise corresponds in time to the detection of the
ester in the culture medium. The pH of the alcohol supplemented media
dropped to approximately 6.8 sometime after 72 hours. This may reflect
50
-------
\J}
EtOH
COLUMN« 5', 15% ETHYLENE GLYCOL
SUCCINATE ON GAS CHROM-P
TEMP. COLUMN 45 C, INJ. I65C, DEI I65C
CARRIER •' N2
H2 FLAME
2/iL (SAMPLE)
UNKNOWN (ETHYL BUTYRATE)
ORGANIN (ACIDS)
90 180 270 360 450 540
TIME (SEC)
630 720
Figure 11. Gas-liquid-chromatograph (GLC) of culture supernatant in presence and
absence of ethyl alcohol
-------
B
EtOH
UNKNOWN
UNKNOWN
REINFORCED
WITH
ETHYL BUTYRATE
90
180
0
90
180
Ld
Q_
UJ
LU
tr
EtOH
ETHYL BUTYRATE
0
90
180 0
TIME (SEC)
90
180
Figure 12.
GLC of Aquacide-concentrated culture supernatant
in presence and absence of ethyl alcohol
52
-------
H-
S2
LU
X
^
s
Q_
UJ
o:
EtOH
UNKNOWN
EtOH
B
UNKNOWN
REINFORCED
WITH
ETHYL BUTYRATE (1=10)
0
90
180 0 90
TIME (SEC)
180
Figure 13. GLC of ether extract of culture supernatant in
presence and absence of ethyl butyrate
53
-------
(A) MEDIUM 3E (Arg) + EtOH (2%)
(B) MEDIUM I (Arg)
(C) MEDIUM I (PPYE) + EtOH(2%)
(D) MEDIUM I (PPYE)
48
TIME (HOURS)
Figure Ik.
pH changes in ethyl alcohol supplemented and
nonsupplemented culture media during growth of
Z. ramigera 115
the lowering of the alcohol content of the medium and a loss of the
already formed ester "by volatization. Growth at this point is in the
late stationary to early decline phase.
Ethyl butyrate was produced by cultures in ethyl alcohol supplemented
media only in the temperature range of lk° to 37°C, with an optimum of
28°C. Z. ramigera could grow (multiply) over a range of k° to UO°C.
Transfer of cells grown at ^°C in alcohol media to 28°C resulted in ester
production.
Transfer of cells grown in alcohol free media for extended time periods
(at least one month) to fresh alcohol-supplemented media did not immedi-
ately yield ester formation. However, after several transfers of the
cultures in alcohol supplemented media, the ester was again detected.
Examination of cell-free culture supernates for the ability to produce
ethyl butyrate suggests the presence of a heat labile factor free in the
medium which has a role in ester synthesis. Table 11 illustrates the
effects of heat on ester production above the levels formed during
cultur60 Ethyl alcohol was added to the supernate to bring the alcohol
-------
concentration to 2% (v/v) as needed. Table 11 shows that the unheated
supernate system yielded small increases in ester concentration.
TABLE 11. Ester production in culture supernates which had been
heated at various temperatures and resupplemented with
2% ethyl alcohol showing that cultures which had been
grown in the presence of ethyl alcohol produced a heat
labile substance which was associated with ester pro-
duction whereas cells which had not been grown in the
presence of alcohol produced no ester
Holding
Temp. CC
Growth supplemented
121°C 100°C 60°C 45°C Ua
Growth not supplemented
121°C 100°C 60°C 4S°C U
0
4
20
28
37
aU = unheated supernate
Addition of unwashed or washed alcohol-grown cells to filter sterilized
EFYE culture supernates, heated to 45° or 60°C did not result in the
formation of ethyl butyrate. However, addition of these cells to heated
PPYE culture filtrate supplemented with 2% ethyl alcohol resulted in the
formation of the ester with a maximum yield of 0.5%. Addition of large
quantities of these cells to phosphate buffer, pH 7.2, containing 2%
alcohol resulted in the formation of the ester in very low concentrations
(less than 0.1%); in this case the ester was lost very rapidly, probably
due to volatization. Non-alcohol grown cells did not cause ester forma-
tion under similar conditions.
The ability of Zoogloea ramigera 115 to grow on organic acids and alco-
hols had been demonstrated (5b). Further, the organism is known to pro-
duce poly-beta-hydroxybutric acid granules which are observed to be
depleted when the organism (27, 73) is placed in a starvation system.
This implies that short chain acids, including butyric acid, are being
formed.
55
-------
During the growth of Z_. ramigera in alcohol free media, no esters were
detected, either by gas chromatography or examination of an alcoholic
extract of the culture supernate by infrared spectroscopy. Addition of
alcohol to the medium, however, resulted in the production of esters by
the organism. Ethyl butyrate was identified as the only detectable ester
formed during culture in ethyl alcohol.
Formation of the ester is catalyzed by a heat-labile factor(s) found in
either the culture medium or cells. This factor esterifies butyric acid
produced as a metabolic by-product and residual ethyl alcohol in the
medium. The catalytic agent is characteristic of an enzyme in its heat
lability, functional temperature, and optimal pH, although no isolation
or purification has been made.
We believe this esterification procedure may permit the rapid removal
of acids which are formed as metabolic by-products and may explain why
the organism has been reported not to have an acid reaction on organic
substrates. Organic acids and alcohols present in waste waters would
be in low concentration, but the supply would be omnipresent due to the
metabolic activities of other microorganisms. Removal of acid products
could be of survival value to the organisms. We have observed the for-
mation of ethyl butyrate in a mixed culture system containing Z. ramigera
115 and a saccharolytic yeast (unpublished data), suggesting that the
ethyl alcohol formed by the yeast was being esterified by the bacterium.
Although no data are presented, there is no a priori reason why other
alcohols would not also be esterified if present in the growth medium.
This would be the case in sewage.
Furthermore, Z_. ramigera is suspected of being related to Acetomonas
and Acetobacter, organisms associated with C2 metabolism. We believe
this esterifying type of metabolic activity could be responsible for
the formation of bouquets in wines and vinegars and also aids in con-
verting undesirable sewage odors to jLess objectionable odors.
Antigenic Relationships Among the Floe-Forming Bacteria
Immunodiffusion reactions between rabbit antisera and trichloroactic
acid soluble (polysaccharide, carbohydrate) antigens are summarized in
Table 12. Antisera to Z. ramigera 115 reacted with other Zoogloea
isolates, the C-l and C-3 organisms and Acetobacter suboxydans. No
reaction with Acetobacter oxydans or the Pseudomonas isolates tested
could be demonstrated.
Zoogloea I-16-M antisera also reacted with the other Zoogloea isolates.
However, no reaction with A. suboxydans could be demonstrated; whereas
it did react with A. oxydans and the Pseudomonas isolates tested.
Of the antisera tested, none had identical reaction patterns. This is
expected because each organism had been considered on the basis of bio-
chemical and morphological examination to be different organisms. The
extensive amount of reaction demonstrated among the isolates is indicative
56
-------
TABLE 12. Summary of immunodiffusion reactions
Antisera
Antigens
(TCA extracts)
115
Z. ramigera
I-16-M
Z. ramigera
P-8-4
Z. Fillipendula
C-3
G(-) rod
ATCC 621
A. suboxydans
ATCC 6433
A. oxydans
C-l
G(-) rod
C-22-4
Pseudomonas sp.
ATCC 13867
Ps. denitrificans
^
\
Z. 115
+
+wk
+wk
+
+ C2F)
-
•*•
-
Z. I-16-M
+
•f
+wk
+
-
+wk
-
+
+
Z. P-8-4
+
+
+
+wk
•»-wk
-
-
M
+
C-3
•f
+wk
•*
+
-
+wk
+
—
621
A. sub
+ (2F)
+wk
+wk
-
+
•»•
6433
A. ox
-
+
-
.
+
•»•
»
+ = immunodif fusion reaction observed
- = immunodiffusion reaction not present
2F = 2 fused lines; i.e., lines of identity
-------
of phylogenetic relationship among them. Typical reaction patterns are
presented in Fig. 15.
Also of particular significance are the strong double lines of identity
between A. suboxydans and Zoogloea 115. These are clearly shown in
Figs. 15A and 15B. This suggests that Zoogloea ramigera 115 is closely
related to A. suboxydans - perhaps more closely related than A. suboxydans
and A. oxydans . However, biochemical and morphological data indicate
that Z. 115 and A. suboxydans are not identical.
Reference to Table 12 indicates that antisera to C-l, C-22-k and ATCC
13867 were not prepared nor were antigens to these organisms tested
against A. suboxydans or A. oxydans. Reciprocal reactions were carried
out with all other isolates. The only reciprocal reaction which could
not reaffirm its counterpart reaction was with C-3 and A. oxydans; i.e.
C-3 antisera reacted weakly with A. oxydans antigen but A. oxydans anti-
sera did not react with C-3 antigen.
Deoxyribonucleic acid (DNA) density and mole f0 G/C
DNA densities were calculated from the position of E. coli reference DNA
(1.710 density and 51% G/C) by the following equation:
(density) p = po + U.2W2 r2 - r§ x 10~10 g cm3
where :
po = density of reference DNA
w = speed of ultracentrifuge rotation in radians sec"1
ro = distance of reference DNA from center of rotation
r = distance of sample DNA from center of rotation
Mole % of G/C was calculated from the following equation:
p - 1.660
0.098
x 100
Values are presented in Table 13 and indicate that all of the organisms
with the exception of A. oxydans and Z. filipendula P-8-U had GC ratios
in the range of 60 to o"^. This supports the data indicating immunologic
relatedness although it is surprising that A. oxydans and Z. filipendula
P-8-^ had considerably lower GC ratios.
Smooth-rough colony variants of Z. ramigera 115 have been observed on
agar plate cultures. Also we have observed vastly different cell mor-
phology with 115 when cultivated in the cold. Consequently DNA analyses
were made on smooth, rough and 5°C cultivated organisms in addition to
normal shake flask grown cells. Each is listed separately in Table 13-
Comparisons of isolates must be made in conjunction of other types of
data previously reported (e.g. biochemical, immunological).
-------
1. A. suboxydans
2. Z. ramigera 115
3. A. oxydans
U. Z. filipendula P-8-U
5. Z. ramigera I-16-M
-1. A. suboxydans antiserum
1. A. suboxydans
2. Z. ramigera 115
6. C-3
U. Z. filipendula P-8-U
5. Z. ramigera I-16-M
-1. A. suboxydans antiserum
2. Z. ramigera 115
U. Z. filipendula P-8-U
5. Z. ramigera I-16-M
-U. Z. filipendula P-8-U
antiserum
€)©©
2. Z. ramigera 115
5. Z. ramigera I-16-M
C-22-U
7.
-5.
Z. ramigera I-16-M
antiserum
Figure 15.
Photographs of typical irmnunodiffusion reactions
between rabbit antisera (center well) and trichloro-
acetic acid (TCA) antigens from floe-formers
59
-------
TABLE 13. Summary of deoxyribonucleic acid (DNA) density and
base ratio (G/C) analysis
cr\
o
Isolate Density relative
to E_. coli DNA
Zoogloea r ami g era 115
Zoogloea ramigera 115 (rough)
Zoogloea ramigera 115 (smooth)
Zoogloea ramigera 115 (5°C)
Zoogloea ramigera I-16-M
Zoogloea filipendula P-8-4
Pseudomonas denitrificans P-95-5
Pseudomonas denitrificans ATCC 13867
Acetobacter suboxydans ATCC 621
Acetobacter oxydans ATCC 6433
C-l
C-3
C-22-4
Escherichia coli standard
1.7224
1.7199
1.7226
1.7225
1.7232
*
1.7222
1.7235
1.7188
*
1 . 7206
1.7195
1.7211
1.710
mole %
(luanine + cytosine
(C/C ratio)
63.7
61.1
63.9
63.8
64.5
51*
63.5
64.8
60.1
51*
61.8
60.7
62.3
51
* could not be differentiated from E. coli DNA standard, 1.710-= 51% GC
-------
All floe-forming isolates examined via inmrunodiffusion analysis have
not been analyzed by nucleic acid density (% G/C). This is because the
two studies were carried out at different times and each procedure is
fraught with different types of experimental difficulty. For example,
the antibody stimulating capacity of different isolates varies consider-
ably and in some cases required that the immunization of animals be
repeated with considerable delay in time. We ultimately used Freunds
complete adjuvant to stimulate antigenic response in the rabbits. Also,
in the case of nucleic acid analysis the procedure of Marmur had to be
modified with some isolates because the copious amount of polysaccharide
produced by the cells either adsorbed salts from the reagent or adsorbed
DNA.
Complete profiles on all of the isolates must await further experimenta-
tion.
It is perhaps worth mentioning that we have conducted a computer taxo-
nomic analysis and have compared 22 isolates of Zoogloea and floe-forming
pseudomonads to 3l8 bacteria isolated from Lake Erie over a three year
period. The interesting result of this analysis is that all 22 isolates
clustered at a 90-100$ similarity level and suggests a strong relation-
ship among all of the floe-forming isolates examined.
The combined immunologic, GC ratio and computer data are strongly sug-
gestive of a close phylogenetic relationship among the floe-forming
bacteria classified as Zoogloea3 Acetobacter and Pseudomonas and that
many bacteria within this related group appear to be more closely
related to each other than they are to other species within the genera
in which they are currently placed.
Structure and Composition of Polymer Surrounding Z. ramigera 115
As mentioned previously the zoogloeal matrix which has historically been
described as a finger-like projection (10, lU, 33) can be seen in the
photomicrographs of isolate 115 shown in Figs. l£-l, 16-2, and 16-3.
Figure 16-U is a phase-contrast photomicrograph of individual packets of
isolate 115 cells. We have previously reported that the finger-like
projections appear to consist of individual packets of cells (UO). The
arrows in Fig. 16-3 point to interfaces between individual cell packets
which have been stained using dilute crystal violet.
Examination of the zoogloeal floes in the native state using the freeze-
etching technique and subsequent electron microscopy revealed the floe
structure shown in Fig. 17- Matrix structure resembling or analogous
to the individual packet structure shown in Fig. 16-^ can be observed;
an interface (l) between two floe packets can also be seen. Cells
embedded within the matrix are not readily observed in Fig. 17-1; how-
ever, Fig. 17-2 clearly shows the presence of a cell (C) embedded within
the matrix. The matrix appears to consist of polymer which forms a
network of fibrous strands (s) composed of two units of different diame-
ter, one measuring i*0-60nm and the other U-5nm. Figure 18-1 illustrates
exocellular strands adhering to cells of isolate 115.
61
-------
J
~ .. • ••
Figure 16 (l). Z. ramigera 115 fingerlike projections composed
of cells embedded within the zoogloeal matrix.
Cells were suspended in k2% bovine serum albumin
(2). Z. ramigera 115 floes stained with 1.0$ crystal
violet
(3).
Z. ramigera 115 floes stained with crystal violet,
showing interfaces (I) between individual clumps
of cells
Phase-contrast photomicrograph of Z. ramigera 115
cells stained by Maneval's method and showing
individual packets of cells. Each packet is
surrounded by capsular or zoogloea matrix
-------
•
v»
-rjti
v^ .^^?^|fe H ~2£^" •
--;-^^% -^ .O^£^
^ - - .\: r-" ^:?^-<
•-•
0
Figure 17. Matrix of Zoogloea r_ajni"era 115. (1) Freeze-etching of 115 showing
cell-floes (F). Cells (C) vrithin the floes are present. Inter-
faces (1) are present between the cell-floes. XU,000 (2) Freeze-
etching of single cell (C) embedded within zoogloeal matrix.
Strands (S) are present around the cells; the thicker strands are
1+00-600 A, while the thinner strands axe UO-50 A. X50,000
63
-------
c^ i
Figure 18.
Comparison of strands of Zoogloea ramigera 115 "by
freeze-etching and negative staining.(l) 115
surrounded by strands (S) of exocellular matrix.
The cytoplasm (Cy) may be seen; several free cells
are also present. Xll,300 (2) Negative stain
of matrix surrounding 115 cells. X^2,000
(3) Another negative stain of matrix. Strands (S)
are present and measure 60-130 A XU2,000
-------
The floe matrix has also been examined after negative staining. This
technique (Fig. 18-2, 18-3) also reveals strands resembling those
observed in frozen-etched specimens. The strands in this micrograph
are 6-13nm in diameter.
Figures 19-1 and 19-2 are thin sections of zoogloeal floes -which have
been embedded and stained with lead citrate. The extracellular strands
can be detected as branching fibrils with a diameter of k - 6 nm.
Figure 19-2 shows void space (v) surrounding cells embedded in the
matrix. This could result from motility within the floe, from lack of
polymer synthesis at this location on the cell surface, or it may con-
sist of substances undetectable using this technique. A characteristic
gram negative cell wall is present and the cell appears to be packed
with ribosomes.
Purified extracellular matrix material has also been examined with the
electron microscope and is shown in Fig. 20. Polymer strands resembling
those shown in the native state (Figs. 17, 18, 19) can be seen and meas-
ure approximately 2 - 5 nm in diameter. An infrared absorption spectrum
of the purified material shown in Fig. 20 is presented in Fig. 21. The
following functional group assignments were made: OH, 2.95 urn; C-H,
3.38 - 3.^4- urn; C=0 of ionized carboxyl or aldehyde, 6.15 urn and 7.1
weak C=0 of aldehyde, 5-8 um and 7-3 um; tertiary CH-OH, 8.65 um; band
typical of cellulose or polysaccharide ring, 9.k pm. The I-R spectrum
is identified as that of a polysaccharide which may contain carboxyl
functions or terminal aldehyde groups.
Chromatograms of polymer hydrolysate (HC1 or H2S04) revealed a single
spot when sprayed with either aniline-diphenylamine or aniline-acid-
oxalate. These reagents developed spots which had an Rf value identical
to glucose in the solvent systems employed. The spot color was also
identical to that of glucose. It was therefore concluded that glucose
is the primary and possibly the only constituent of the polymer. How-
ever, subsequent examinations revealed the presence of galactose in the
polymer. Several ninhydrin positive spots were present in crude polymer
extracts but disappeared during the polymer purification procedure and
this was considered evidence of polymer purity. Although the I-R
spectrum was interpreted as possibly containing carboxyl groups, no
uronic or other organic acids could be demonstrated.
The polymer was not susceptible to hydrolysis by either alpha or beta
amylase under the described conditions. The polymer and whole 115 cell-
floes repeatedly gave significant positive responses to cellulase in
comparison to carboxymethylcellulose controls.
The only other available Z. ramigera (isolate I-16-M from Crabtree and
McCoy) (26, 27) culture has been examined for comparative purposes.
Figures 22-1 and 22-2 are shadow-case preparations of cells which were
treated with 1 N NaOH for 20 minutes at 100°C (72). An extracellular
polymer is present which appears distinctly different from that produced
65
-------
Cw
0
PP
©
Ficure 19.
Thin sections of Zoorloea ramigera 115. (l) Thin section of 115
showing cell wall (Cw), cell n.einbrane (Cm), ribosomes (R), and
strands (S) of exocellular matrix. X6U,000 (2) Thin section
of 115. In addition to the above a polyphosphate granule (FP)
and a distinct void (V) are present. The convoluted wall of 115
is quite in evidence. The diameter of the strands is kO-60 A.
X6?,000 Thin sections and photographs courtesy of Dr. R.M. Pfister
66
-------
•
' -4r\*:
. •» «^ ^ ^.'
v, •%- •••«•'
-«>* <^ Vti
• '^fc^t \ *m
^k e "' *S !
Figure 20. Purified extract of Zoogloea ramigera 115 exocellular
matrix. Strands present are 20-50 A in diameter.
X122,500 Photograph courtesy of Dr. R.M. Pfister
67
-------
MOONS
MICRONS
CO
4000
Figure 21. Infrared spectrum of purified exocellular matrix of Zoogloea
ramigera 115
-------
- • <*g
N ~ A ' - ^--"^V
;\;: ; v^ r^^srx
>-^ - ^»^o^
- c*s
~-^- is.
.~
3^*^*. .-^**VS
S ^ -
®
Figure 22. Comparison of Zoogloea ramigera I-16-M to 115
after treatment with NaOH.(l) Shadow-casting
of I-16-M showing fibrils (Fb) and flattened
cells (C), X19,500. (2) Another shadow-casting
of I-16-M. Single fibrils (Fs) are 120-1^0 A in
diameter, X22,?00. (3) Shadow-casting of 115
showing remnants of cells (C). Dark granules are
probably poly-beta-hydroxy-butyric acid (PHB)
XUl,200
69
-------
by isolate 115 when treated in the same manner. No shadow-cast photo-
graphs of isolate 115 cells are presented because the 115 polymer is
soluble in IN NaOH. Figures 23-1 and 23-2 are frozen-etched prepara-
tions of the I-16-M isolate. Distinct extracellular strands can be seen
attached to cells but the polymer configuration is different from that
shown for 115 cells in Figs. 17 and 18.
The larger fibrils shown in Fig. 22 are approximately 12 to Ik nm in
diameter and appear to be formed from elementary fibrils which are 2-5
nm in diameter. The large fibrils shown in Fig. 23 are about ^0 nm in
diameter. This suggested that in frozen etched preparations, material
adheres to the fibrils, whereas it is removed when treated with IN NaOH
(Fig. 22). A granule which is presumed to be PHB is also present.
Figure 2U-1 shows PHB granules isolated from 115 cells and Fig. 2*4-2
shows more clearly the PHB granules in I-16-M cells.
Figure 25 compares individual cells and floes of Z. rajnigera I-16-M
under brightfield and ultraviolet illumination.
Z. rajnigera isolate I-16-M has been described as a nonzoogloeal matrix
producing organism (27). Figure 7a is a photograph of an isolate I-16-M
floe which has been stained with 1% aqueous crystal violet. No exocellu-
lar material was observed using this technique and many of the cells
appeared to be free from the main clump of cells. When the isolate
I-16-M floes were examined in a wet mount without staining they also
appeared as dispersed cells without an identifiable extracellular matrix
as shown in Fig. 25-1. However, Fig. 25-2 shows that when the same floes
as shown in Fig. 25-1 are stained with the fluorescent dye Paper White BP
and photographed through an ultraviolet microscope, the dye can be seen
to be concentrated between the cells. This suggested the presence of
substances which have a high affinity for the dye (e.g., cellulose or
orther polysaccharides). Figures 25-3? 25-^, 25-5 and 25-6 show the same
effect using larger floes and lower magnification.
The association of the fluorescent dye with cell-floes was quite rapid
and wet mounts could be examined immediately after preparation. Fluores-
cence with I-16-M was initially quite intense, but gradually lessened
during a 5 minute interval. Z. ramigera 115 fluoresced weakly when com-
pared to I-16-M or particles of cellulose used as controls.
Several other isolates which have been partially described previously
were also examined for the presence of extracellular polymers. Pseudo-
monas C-3 was examined by several techniques (Fig. 26) all of which
showed the presence of fibrils surrounding the cells. Figure 26-3 is a
shadow cast preparation and shows fibrils obtained by placing the cell-
floes in IN NaOH for 2k hours at 28°C. Ghosts of several cells remain.
Fibrils extend outwardly (Fig. 26-1) as rays (f) from the cross sections.
These rays may be analogous to the polymer matrix observed with frozen
etched preparations of Z. ramigera isolate 115 and may represent a micro-
capsule surrounding the cells.
TO
-------
Figure 23. Fibrils of Zoogloea ramigera I-16-M. (l) Freeze-etching
showing fibrils (F) around I-16-M. The cell wall(Cw),
cytoplasmic membrane (Cm), and cytoplasm (Cy) are also
present. X^8,000 (2) Freeze-etching of I-16-M showing
additional fibrils 7^8,000
71
-------
Me
lu
>v
^
Figure 2U. Poly-beta-hydroxybutyric acid granules (PHB) obtained from Zoogloea ramigera 115
and I-16-M. (l) Carbon replica of PHB granules obtained from freeze-dried prepara-
tion of Zoogloea ranigera 115. Granules possess a rough surface and perhaps a
partial membrane covering (Me). X19,^00 (2) Freeze-etching of Zoogloea ramigera
I-16-M showing stretched PHB granules. The cell wall (Cw) and cytoplasm (Cy) are
also visible X8l,000
72
-------
lOu
IQu IQu
Figure 25.
IQOu
Brightfield and ultraviolet microscopy of Zoogloea
ramigera I-16-M. (l) Individual cells viewed with
white light, X2,000. (2) Individual cells viewed
with ultraviolet light, X2,000. (3) Floe viewed
with white light, X2,200. (U) Same floe viewed
with ultraviolet light, X2,200. (5) and (6) other
floes viewed with ultraviolet light, (5) X2^0 and
(6) X226. All cells and cell-floes were photo-
graphed in the presence of Paper White BP, a fluor-
escent brightener
73
-------
Figure 26.
:.^K^
r V-.S*:
Freeze-etchings, shadow-casting, and carbon replica
of Pseudomonas C-3. (l) Cross section as seen with
freeze-etching. Rays (r) extending outward and cyto-
plasm (cy) are seen, X25,800. (2) Freeze-etching
showing fibrils (f). In addition the cell wall (cw),
cell membrane (cm), and cytoplasm (cy) are visible,
X25,200. (3) Shadow-casting showing fibrils (f) and
granules, probably PHB. The remnants of a cell (c)
are seen, X39>900« (*0 Carbon replica showing cells
(c) and fibrils (f) intertwining around cells. Thicker
fibrils, i.e., flagella (fl), are present, X9,000
-------
Figure 26 shows an entire cell with elementary fibrils (f) adhering to
the outer cell surface. The cell wall (cw), cell membrane (cm), and
cytoplasm (cy) can be seen.
Figure 26-k is a carbon replica of isolate C-3. Fibrils are quite com-
mon and crisscross over the cells. Thicker fibrils which may be flag-
ella (fl) can be seen. Figures 26-1 and 26-2 are frozen etched prepa-
rations of isolate C-3.
Shadow cast preparations of other floe-forming bacteria also showed the
presence of fibrils attached to cells. Figure 27-1 is a preparation of
Z. filipendula isolate P-8-U (26, 27, Ul). Figure 27-2 is Fseudomonas
denitrificans isolate P-95-5 (26, 27, kl). Figure 27*3 is an unidenti-
fied gram negative rod isolate C-22-4 (26, 27, Ul). Figure 27-k is a
gram negative rod which had been identified as a Z. ra.Tni.gera isolate
Z-SC-38 (MO.
The zoogloeal matrix which has been the primary basis for differentiating
Zoogloea from other pseudomonads is composed of polymer strands. The
"finger-like" projections appear to be built up from generally globular
shaped packets of cells which adhere one to another. This is interpreted
as being responsible for the flocculent habit of growth of this particu-
lar organism. The involvement of poly-beta-hydroxybutyric acid (PHB) as
reported by Crabtree et al. (27) has not been ruled out but does not
appear to play a direct role. This will be considered further in sub-
sequent section of this report. Both 115 and I-16-M isolates produce
large amounts of PHB under certain growth conditions.
Two sizes of polymer strands are shown in Figs. 17 and 18. The purified
polymer (Fig. 20) has only one size strand which measures 2 to 6 nm in
diameter. This suggests that the larger strand shown in Fig. 17 may
have been induced by the formation of ice crystals during the freeze-
etching procedure. The loss of pure water during sublimation could
cause a concentration of the residual solution, leaving a eutectic mix-
ture remaining between ice crystals. This mixture would tend to associ-
ate with the grna.11 strands causing them to appear larger.
The available chemical and spectroscopic data indicate that the polymer
of isolate 115 is a polysaccharide; it is slightly soluble in water and
solubility increases in alkaline solutions (NH4OH < NaQH). Glucose and
galactose are the only polymer components obtained from acid hydrolysis
and susceptibility of the polymer to ceHulase strongly suggests that
the polymer resembles cellulose (i.e. contains 1, U-beta-glycosidic
bonds). Identification of the polymer as a type of cellulose must be
made with caution because the crude cellulase enzyme may contain an
unknown activity on the polysaccharide substrate. The polymer does not
appear to be identical to the bacterial cellulose described by Muhlethaler
(69) but has some resemblance to the cellulose fibrils shown by Gibson
and Colvin (^5). Further supporting evidence is being sought. Lack of
susceptibility to either alpha or beta amylase indicates an absence of 1,
^-alpha-glycosidic bonds. A negative color response to iodine is also
noted.
75
-------
Figure 27.
Shadow- cast ing of four floe -forming bacteria, (l) Zoogloea
filipendula P-8-U, X21,000 (2) Pseudomonas dentrificans
P-95-5, X23,500. (3) unidentified gram-negative rod C-22-U,
Xl6,200. (U) Zoogloea ramigera Z-SC-38, X9,200. Cells (C)
and fibrils are seen.
76
-------
The exopolymer which surrounds the I-16-M isolate has not been isolated
and purified so that comparisons to the polymer of isolate 115 must be
made with caution. However, the difference in fine structure as well as
different solubility properties in basic solutions suggests that the
polymers are not identical, although the polymers from both 115 and I-16-M
cells are susceptible to cellulase.
The polymer of isolate 115 appears to act as a poly electrolyte, analo-
gous to synthetic ion exchange resins or those used in gel filtration.
It is probable that the matrix can serve a nutritional function for the
cell by concentrating nutrients from solution. This could explain how
organisms growing in extremely dilute nutritional aquatic habitats can
accumulate essential nutrients. These same suggestions are applicable
to the I-16-M isolate, which we conclude is a distinctly different organ-
ism than Z_. rairrigera isolate 115-
The possibility of cellulose synthesis by cells which do not produce
acids raises some interesting taxonomic problems. If the generality of
this result can be established it may be a definitive criterion which
will aid in identifying organisms in the genus Zoogloea.
The data presented suggest that bacteria which have been isolated on the
basis of their characteristic flocculent growth habit, all possess extra-
cellular fibrillar polymers. Entanglement of cells among fibrils, or
adsorption of cells to fibrils is a plausible explanation of the floccu-
lation phenomenon. This conclusion supports the views of Tenney and
Stumm (90), Busch and Stumm (13), Clark (23) and to some extent those
of Finstein (38) and Heukelekian (50). The physical and chemical proper-
ties of the specific exocellular fibril polymers will determine the
extent to which water is bound to the polymer and will also determine
the solubility properties of the polymers and their affinity for stain.
Therefore some bacterial polymers will appear as observable capsules
whereas others will not.
The purified polymer from_Z. ramigera isolate 115 was reported to have
similar properties to synthetic polyelectrolytes (U2) and flocculation
of bacteria by synthetic polyelectrolytes would be analogous to the natu-
rally occurring process. It can be further postulated that polyvalent
ions can complex with functional groups on two different elementary
fibrils thereby resulting in an effective bridge and alteration of the
polymer charge. The association of extracellular polymers produced by one
of the isolates (C-22-U) with sub-microscopic inorganic particles has
been reported (?6, See Fig. ^6}. We wish to point out the analogy of
polymer bridges among bacterial cells.to similar polymer bridges among
inorganic or synthetic materials (e.g. polystyrene latex balls) such as
reported by Ries and Meyers (83)•
Floe formation from other types of polymer should also be considered.
Wessman and Miller (99) found that clumping of Pasteurella pestis was
caused by the polymerization of extracellular nucleic acids that were
excreted by the cells. The role of 7KB reported by Crabtree et al. (26)
77
-------
should also be further considered as an explanation of bacterial floc-
culation. (Also see section on polymer production and Fig. Ul.)
One additional point concerns the taxonomic status of the floe-forming
isolates described. All of the floes were susceptible to cellulase and
showed a positive hexose response within 15 to 120 min. Quantitative
values are not presented here because the polymers were not always
obtained in pure form and the quantitative variations probably reflect
varying amounts of polymer surrounding individual cell isolates.
All of the isolates are polarity flagellated rods which do not resemble
Acetobacter xylinum or Sarcina ventriculi. Few of the isolates are acid
producers and most do not closely resemble Grluconobacter or Acetomonas
in this regard. If the polymer around certain isolates proves to be
cellulose, a taxonomic reappraisal would be in order.
Aquatic floe-forming organisms other than those investigated during this
study have since been shown, in this laboratory, to produce extracellular
polymer fibrils (e.g. Azotobacter, Bacillus, Chromobacterium, Flavo-
bacterium) and the association of floe-format ion with extracellular
fibrils appears to be a valid generalization. Figure 28 is an electron
micrograph of a frozen etched preparation of a gram positive spore form-
ing aerobic rod (Bacillus) and serves to illustrate that bioflocculation
is not limited to gram negative organisms. See also (3^> 59)'
One additional comment which should be made is that some bacteria tend
to grow as a pellicle at an air-liquid interface. These surface films
of bacteria may then settle and appear as floes. It is probably without
value to differentiate between pellicle formers and floe-formers in a
discussion of bioflocculation since the net effect is quite similar.
Figures 29A and 29B are electron micrographs of carbon replicas of an
isolate C-3 pellicle which shows the close packing of cells that grow
at an air-water interface.
Concentration and Accumulation of Chemicals
Gross uptake of metallic ions. Results of these uptake studies are
listed in Table 14.These experiments gave an indication of the extent
which pregrown cells would concentrate and accumulate metal ions from
solution in a non-growth situation. The 115 cell-floes have a remarkable
high affinity for Co"1"2, Cu+2, and Fe+3, and somewhat less for Ni+2.
Values obtained after contact periods of 2 and 3 days indicate that no
further uptake of ions occurs beyond 18 hours. No data were obtained
for time intervals less than 18 hours in this experiment. Comparison
of data obtained using 115 cells to data from I-16-M cells shows that
approximately twice as much metal ion is accumulated by the zoogloeal
producing strain. This suggests that about half the ion uptake is con-
centrated in the zoogloeal matrix surrounding the cells if it is assumed
that 115 has a matrix and I-16-M has none. However, Figs. 22, 23 and 25
illustrated a matrix around I-16-M but the extent has not been quantitated.
78
-------
Figure 28. Electromicrograph of a frozen etched preparation of a gram positive Bacillus species
showing that bioflocculation is not limited to gram negative organisms. X37,000
-------
Figure 29.
Electron micrographs of carbon replicas of surface
pellicles of isolate C-3 shovrLng closely adhering
cells; A, X30,000, B, X50,000
80
-------
TABLE lk. Values showing initial concentration of metallic ion in solution ((ig/ml) and
concentration after shaking for 18 hours in presence of Zoogloea rajni gera 115
(0.1 g, dry wt) or Zoogloea r»"" gera I-16-M (0.12 g, dry wt)
Cobalt as Copper as Iron as Nickel as
CuCl2»2H20 FeCl^HjjO NiCl2«6H20
Initial Concentration
Zoogloea ramigera 115 50 240 500 90 400 800 34 180 350 50 250 500
Concentration after
18 hours 1.3 2.2 3.5 73 97 121 0.5 3-2 4.3 41 190 250
i-1 % removal 97.5 99.1 99. 3 18.9 75.8 84.9 98.5 98.2 98.8 18 24 50
Total removed, ir.g/100 mg
cells (dry wt) 2.4 11.9 24.8 0.8 15.1 34 16.7 8.8 17.3 0.4 3 12.5
Zoogloea ramigera I-16-M
Concentration after
18 hours 2.8 64 310 4.7 150" 420 0.3 1.1 187 32.6 164 376
% removal 94.5 73-3 38 94.8 62.5 47. 5 99.1 99.4 46.6 34.8 34.4 24.8
Total removed, mg/100 mg
cans (dry wt) 2.3 8.8 9.5 ^.3 12.5 19 16.8 9 8.2 0.9 4.3 6.2
-------
The uptake of various combinations of cations by Z. ramigera, 115 was also
studied. As shown in Table Ik, almost 100% of Co+2 and Fe+3 were absorbed
by 115 after an 18 hour contact period. Similar experiments calculated
in micromoles (nmoles) give analogous results (Table 15) . The theoretical
value damoles added) is the sum of all cations placed in solution for a
particular test. The number of nmoles removed from solution is the quan-
tity removed by the cell-floes. When two or three ions were added
(Tables 16 and 17), the greatest accumulation usually occurred in the
second concentration of each complete set of ions. Although the first
concentration of each set of ions might be thought to absorb all of the
ions present because of the availability of sites, the low values suggest
this is unlikely. The decrease in values obtained with the third concen-
tration suggests a loss from oversaturation. The order of absorption is
Fe+3> Co+2> Cu+2> Ni+2.
Uptake of metal ions by I-l£-M indicates that cells in the absence of
zoogloeal matrix can concentrate a considerable amount of metal ion. How-
ever, the ions may accumulate, at least in part, on the polymer strands
shown to be present around I-16-M cells.
The cells accumulated 3k% of their total weight of Cu+2 and approximately
25% of Co+2. This precludes use of this type of experiment to determine
metal uptake by growing cells vs. time. That is, no accurate cell weight
determinations could be made in the presence of relatively high concentra-
tions of ions. Cell counting techniques are not feasible when the cells
are embedded in a zoogloeal matrix. More precise data pertaining to metal
uptake by both growing and non-growing cells were obtained using the radio-
isotope Zn65Clg.
Accumulation of Zn*"2 by actively growing cells. Figure 30A shows the
accumulation of Zn65 in the presence of 10 (ig carrier Zn+2/ml of growth
by Z_. ramigera cell-floes over an 11 day period. Loss of Zn+2 from cul-
ture medium is also shown. Loss of Zn+2 from supernatant correlates with
uptake of Zn+2 by cell- floes when total counts/min are compared. Accumu-
lation of Zn+2/0.2 g cell-floes has also been plotted. This curve inci-
cates that the rate of Zn+2 uptake is constant with time from the 6th to
the llth day. Total uptake during this time increases because of the net
increase in cell-floe weight. Total weight increase of cell-floe vs. time
is shown in Fig. 30B. Weight of cell-floe is emphasized because cell
numbers do not increase in proportion to weight over the entire 11 day
period. The decrease in Zn+2 associated with the cell-floe fraction
between the 5th and 7th day suggests a shift in metabolism of the organ-
ism. This could possibly reflect either a conversion from cell repro-
duction to zoogloeal matrix synthesis or accumulation and utilization of
poly-beta-hydroxybutyric acid within the cells. The release of Zn65 from
cell-floe back into growth medium during the 5-7 day interval appears to
be real. Similar curves were obtained with Zn65 alone or with Zn65 plus
1 |ig/ml carrier Zn65 plus 1 |ig/ml carrier Zn+2.
82
-------
TABLE 15. Gross uptake of metallic ions after 18 hour contact with
pre-grown Zoogloea ramigera 115 cell-floes
Cations
Series
Theoretical (ymoles added)
ymoles removed from solution
% of theoretical uptake
1
0.63
0.63
100
Fe+3
2
3.15
3.15
100
3
6.30
6.30
100
1
0.85
0.85
100
Co*2
2
4.25
4.25
100
3
8.5
8.5
100
+2
Cations Cu
Series 12 3
Theoretical (nmoles added) 1.26 6,30 12.60
nmoles removed from solution 0.1 *• 4,?8 4.?6 10.53 11.9
% of theoretical uptake 13 0 ?6 ?6 83 95
Cation Ni*2
Series 123
Theoretical (nmoles added) 0.85 4.2? 8.54
roles removed from solution 0.15 0.1? 0.51 1.02 2.39 2.0*4- 4.2? 6.65 6.65
of theoretical uptake 18 20 60 24 56 48 50 ?8 ?8
-------
TABLE 16. Gross uptake of various combinations of metallic ions
(two cations) after 18 hour contact with pre-grown
Zoogloea ramlgera 115 cell-floes
Concen- Cations Theoretical
tration (lomoles added)
Fe+3
1 Ni"1"2
Fe+3+Ni*-2
Fe+3
2 Ni*
Fe+3+Ni+2
Fe+3
«.+2
3ruX n
+3 +2
Fe +Ni
Fe+3
1 Cu+2
Pe*1"3^2
Fe+3
2 Cu+2
Fe+3
3 Cu+2
Co+2
1 Ni"*"2
Co+2+Ni+2
Co+2
2 Ni*1"2
Co"1'2
- MJt+2
j W JL
0.63
0.85
1.48
3.15
4,27
7.42
6.30
8.54
0.63
1.26
1.89
3.15
6.30
9.45
6.30
12,60
18.90
0.85
0.85
1.70
4.25
4J27
8.52
8.50
8.54
17.04
(imoles re- % of theoret-
moved from ical uptake
solution
0.63
0.25
0.88
3.15
4.21
7.36
6.30
1.28
7.58
0.63
0.24
0.87
3.15
6.03
9.18
6.30
5.43
11.73
0.76
0.29
1.05
4.23
3.72
7.95
7.62
—
7.62
100
29
59
100
99
99
100
15
51
100
19
46
100
96
97
100
43
62
89
34
62
99
/N «•••
87
93
90
~
45
-------
TABLE 16. Continued
Concen- Cations
tration
Co+2
1 Fe+3
Co-f2+Fe+3
Co-*"2
2 Fe+3
Co+2+Fe+3
Co+2
3 Fe+3
Co+2+Fe+3
Co*1"2
T ^j^L
Co+2+Cu+2
Co+2
2 Cu+2
Co+2+Cu+2
Co+2
3 Cu
Co+2+Cu+2
Cu+2
1 Co*2
Cu+2+Co+2
Cu+2
2 Co"*"2
Cuh2+Co'*'2
Cu*2
3 Co*1'2
Cu+24Co+2
Theoretical
(pmoles added)
0,85
0.63
1.48
4.25
3.15
7.40
8,50
6.30
14.80
0.85
1.26
2.11
4.25
6.30
10.55
8.50
12.60
21.10
1.26
0.85
2.11
6.30
4.25
10.55
12.60
8.50
21.10
pmoles re-
moved from
0.74
0.63
1.37
3.98
3.15
7.13
1.23
6.30
7,53
0.78
0.18
0.96
4.06
5.66
9.72
4.42
10.22
14.64
0.22
0.82
1.04
4.80
4.15
8.95
12.20
7.82
20.02
% of theoret-
ical uptake
87
100
93
94
100
96
14
100
51
92
14
46
96
90
92
52
81
69
17
97
52
76
98
85
97
92
95
85
-------
TABLE 16. Continued
Concen- Cations Theoretical jimoles re- % of theoret-
tration _ (^moles added) moved from ical uptake
Cu+2 1.26 0.18 14
1 Ni+2 0.85 0.2*4- 28
Cu+2+Ni+2 2.11 0.42 20
Cu+2 6.30 5.^5 8?
2 Ni+2 4.2? 1-25 29
10.57 6.70 64
Cu+2 12.60 9.84 78
Ni+2 8.54
21.14 9.84 47
86
-------
TABLE 17. Gross uptake of various combinations of metallic ions
(three cations) after 18 hour contact with pre-grown
Zoogloea ramlgera 115 cell-floes
Cone en- Cations Theoretical iimoles re-
tration (limoles added) moved from
solution
Co+ii
1 Fe+3
Ni+2
Co+2+Fe+3j-Ni+2
Co+2
2 Fe+3
Ni+2
Co+2+Fe+3+Ni+2
Co+2
3 Fe+3
Ni+2
Co+2+Fe+3HJi+2
Co+2
1 Fe+3
Ni+2
Co+2+Fe+2*-Ni+2
Co+2
2 Fe+3
Ni+2
Co+2+Fe+^Ni+2
Co+2
3 Fe+3
Ni+2
Co+2+Fe+^Ni+2
0.85
0.63
0.85
2.33
4.25
3.15
ll!67
8.50
6.30
8.54
23.34
0.85
0.63
0.85
2.33
4.25
3.15
4.27
11.67
8.50
6.30
8.54
23.34
0.73
0.63
0.34
1.70
4.11
3*. 15
2.90
10.16
2.54
6.30
1.70
10.54
0.75
0.63
0.16
1.54
4.22
3.15
3.58
10.95
8.32
6.30
5.98
20.60
% of theo-
retical
uptake
86
100
40
73
97
100
68
87
30
100
20
45
88
100
19
66
99
100
84
94
98
100
70
88
87
-------
TABLE 17. Continued
Cone en- Cations
tration
Fe+3
1 Ni+2
Cu+2
r e >rWi ^T
Fe+3
2 Ni+2
Cu+2
Fe+3hNi+2+
Fe+3
3 Ni+2
Cu+2
Fe+3-t-Ni+2+
Fe+3
1 Ni+2
Cu+2 ^
Fe+3
2 Ni+2
Cu+2
Fe+3+Ni+2+
Fe+3
3 Ni+2
Cu+2
Fe+3j.Ni+2+
Theoretical
(pjnoles added)
0.63
0.85
1.26
Cu+2 2.74
3.15
4.27
6.30
Cu+2 !3t72
6.30
8.54
12.60
Cu+2 27e/44
0.63
0.85
1.26
Cu+2 2.74
3.15
4.27
6.30
Cu+2 13t?2
6.30
8.54
12.60
Cu+2 27.44
nmoles re-
moved from
solution
0.63
0.42
0.54
1.59
3.15
0.85
5.54
9.54
6.30
0.68
5.71
12.69
0.63
0.16
0.76
1.55
3.15
2.78
5.67
11.60
6.30
2.58
11.40
20.28
% of theo-
retical
uptake
100
49
43
58
100
20
88
70
100
8
45
46
100
19
60
57
100
65
90
85
100
30
90
74
-------
TABLE 17. Continued
Concen- Cations Theoretical nmoles re-
tration (pjnoles added) moved from
solution
Co*2
1 Cu+2
Fe+3
Co+2+Cu+2+Fe+3
Co+2
2 Cu+2
Fe+3
i ^ f^ t *^
Co+2+Cu+2+Fe+3
Co*2
3 Cu+2
F0+3
Co+24Cu+2+Fe+3
Co*2
1 Cu+2
Fe+3
Co+2+Cul'2+Fe+3
Co+2
2 Cu+2
Fe^
Co+SKW^Hre+3
Co+2
3 Cu+2
Fe+3
CO+2+CU+2F6+3
0.85
0.26
0.63
2.74
4.25
6.30
3.15
13.70
8.5
12.6
6.3
27.4
0.85
1.26
0.63
2.74
4.25
6.30
3.15
13.70
8.5
12.6
6.3
27.4
0.73
0.76
0.63
2.12
4.15
5.48
3.15
12.78
6.57
12.20
6.30
25.07
0.71
0.18
0.63
1.52
3.91
3.67
3-15
10.73
2.12
10.22
6.30
18.64
% of theo-
retical
uptake
86
60
100
77
98
87
100
93
77
97
100
92
84
14
100
56
92
58
100
78
25
81
100
68
89
-------
o
o
o
o
O
o
m
O.34
0.30
026
g 0.22
_i_
• oie
«^
o
E 0.14
o
0.10
0.06
B
6
Time,Days
8
10
12
Figure 30. Accumulation of Zn+2 by Zoogloea ramigera 115 during
growth (A) Curve showing counts per minute per 50 ml
of growth medium supernatant vs. time (a); counts per
minute per 0.2 g of cell-floe vs. time (0); counts per
minute per total amount of cell-floe accumulated dur-
ing growth period (A). (B) Total weight of cell-floe
accumulation per 50 ml vs. time
90
-------
Figure 31A shows the accumulation of Zn65 in the presence of 10 ng car-
rier Zn+2/ml of growth medium by Z. rarrrigera I-16-M cell-floes over a
9 day interval. Loss of Zn+2 from culture medium is also shown. Loss
of Zn+2 from supernatant correlates with uptake of Zn+2 by cell-floes
when total counts/min are compared. Total weight increase of cell-floe
vs. time is shown in Fig. 31B.
Counts/min obtained after one day of incubation are not of significance
because of the small quantity of total cell-floe. Therefore, it is not
justifiable to attribute the decrease on the second day to a growth lag.
Total cell-floe weight does not continually rise as with 115 > but recedes
slowly after the second day. The total coun/min/0.2 g cells peaks at
U-5 days. The count rate averages 1000 counts/min higher than 115 under
similar conditions. A comparison with gross uptake studies therfore
indicates that while I-16-M can concentrate Zn+ in a growth-rate uptake
study, massive doses are not absorbed quickly or bound as tightly as
with 115-
Turbidity of the TSB medium containing I-16-M gradually increases after
the second day. The same is true of medium containing 115. The cell-
floes do not, however, lose as much Zn+2 with time as does the 115. In
addition the weight does not increase. Similar curves were obtained with
Zn65 alone or with Zn65 plus 1 (ig/ml carrier Zn+2.
Uptake of Zn4'2 after pregrowth of culture. Figure 32 presents a curve
showing Zn613 accumulation (no carrier) by h day pregrown 115 cell-floes.
Although the uptake curves on 1 day, 3 day, and k day cell-floes were
similar, the absolute values varied. This suggests that the affinity of
cells for metal ions varies with growth state or physiological state of
the organism. For example, 1 day cell-floes showed an average of 1^00
counts/min/0.2 g cells (dry weight) after U to 6 hours; 3 day eell-flocs
had an average of 2750 counts/min/0.2 g cells after the same time. This
indicates that actively growing cells (2^ hour culture) have approximately
half the affinity for Zn+2 accumulation that k day cells have. This is
in general agreement with data presented in Fig. 30A.
Chemical oxygen demand. Figure 33 is a plot of supernatant vs. time of
growth (Standard Methods, I960) and serves to indicate net oxidation of
nutrients by Z. ramigera 115. The curve shows a rapid decrease in the
quantity of oxidizable material during the first two days. A minimum is
reached, followed by a gradual increase in the quantity of oxidizable
material in the supernatant. It might be noted that an inversion of this
curve is somewhat analogous to a growth curve.
An interesting contrast between this graph and the net cell-floe synthe-
sizing curve shown in Fig. 30 is the increase of cell-floe weight shown
in curve B, in contrast to a plateauing of oxidizable materials in this
graph. Results based solely on curve B would indicate that the cells
continue to grow throughout the incubation period. However, the cells
are no longer viable. This might suggest that the constituents in the
medium are already in an oxidized form and could account for lack of
further decrease in the chemical oxygen demand.
91
-------
I04
c
"g ,
I
o
I01
B
10-
10-
4 6
TIME (DAYS)
10
Figure 31.
Accumulation of Zn+2 by Zoogloea ramigera I-16-M
during growth. (A) Curve showing counts per
minute per 50 ml of growth medium supernatant vs.
time (Q)J counts per minute per 0.2 g of cell-
floe vs. time (O). (B) Total weight of cell-floe
accumulation per 50 ml vs. time.
92
-------
£ 82
o
o
I 80
o.
78
CO
E
O
m
I 74
o
o
72
O
o
CM
o
38 r-
36
1>1
34
30
X 28
26
24
B
O
G
I
I
I
2468
Time .Hours
10
Figure 32.
Accumulation of Zrr^ by four day culture of
Zoogloea ramigera 115 cell-floes. (A) Counts
per minute per 50 ml of growth medium super-
natant (X100). (B) Counts per minute per 0.2
g dry weight of cell-floe (X100).
93
-------
28
Time in Days
14
Figure 33.
Chemical oxygen demand of medium containing Zpoglpea
ramigera 115 during a fourteen day interval
-------
Accumulation of Zn+2 at 21° and 28°C. A very rapid uptake of Zn65 plus
10 tag/ml carrier occurred in the presence of k day old Z_. rami gera 115
at "both temperatures (Fig. 3*0 • However, at 28°C there was greater total
uptake. It is observable that the difference in quantity of Zn+2 accumu-
lated is related to the production of zoogloeal matrix. That is, little
or no matrix is synthesized at 21°C and less Zh+2 is accumulated.
Interrelationship between the Zoogloeal matrix and Zn65 absorption.
Similar results were obtained in experiments where Zn013 was added at the
time of inoculation of culture, i.e., zero time, and after 3, ^, or 5
days of growth of the culture. Sixty to seventy percent of the Zn65 label
remained in the supernatant. Approximately 30-1*0$ of the label was
absorbed by the cell-floes. When the cells were separated from matrix
the amount of Zn65 in the matrix represented a three to four-fold concen-
tration over that observed with cells. The decrease of Zn65 in the cell-
floes on day 5 is similar to the decrease noted previously in Fig. 30A.
These results are, summarized in Fig. 35.
Uptake of Zn+2 by a crude extract. Uptake studies were performed with a
crude extract of Z. ramigera 115 exocellular polymer plus Zn65 and either
10 or 100 |ag/ml carrier. A sharp uptake of Zn+2 occurred, followed by a
plateau (Fig. 36). Almost a ten-fold difference exists between the plots
(the lower the quantity of carrier Zn+2, the higher the count rate) which
reinforces the postulation that most of the ion is absorbed by the matrix.
Acid Mine Water
In a series of experiments designed to determine the capacity of polymer
to adsorb Fe+3 from acid mine water, 3^.5 g (wet weight) of cell-floc-gel
was exposed to 50 ml of FeClg solution (125 M-g Fe/ml) and allowed to shake
for 3 hours. The floe was separated from the supernatant and fresh FeCl3
solution was added and the procedure repeated, etc. A total of l£.8 mg
Fe+3 was adsorbed by 3^-5 g (wet weight) of cell-floe which is equivalent
to about 0.3 g dry weight. Other experiments have shown that 90-98% of the
cell-floe weight removed by centrifugation is due to extractable extra-
cellular polysaccharide, after cultures have reached a stationary growth
phase (e.g. Qk hours). This implies that the polysaccharide adsorbs over
100 times its own weight of water. Conversely about 1% of the ceU_-flx>c
wet weight is due to polymer dry weight under experimental conditions.
Table 18 presents data showing adsorption of various mine water ions by
the cell-floe present in 50 ml flasks (average of about 33.0 g wet weight
of cell-floe or 0.30 g dry weight per flask). Approximately 25 to 33% of
all cations assayed were adsorbed by the floes present. Sulfate which is
the predominant anion associated with the cations in mine drainage was
also adsorbed to the extent of 25$. Iron appears to be the predominant
cation present in the mine water (excluding IT1") when viewed on a mg/L.
basis but this is not the case when it is considered on a molar basis.
The following millimolar concentrations were calculated from the mine
water control data in Table 18: Al (6.6), Ca (9), Fe (8.5), Mg (12),
Mn (0.5), Si (l.U), N03 (0.3), and S04 (^7-8). The total anion content
95
-------
10'
8
Time in Hours
10
Figure 34. Accumulation of Zn"5 pius 10 ng/ml carrier by a four-day
culture of Zoogloea ramigera 115 grown at 21 (a) and 28 (®)c
96
-------
100
.9?
.a
jg
1
80
60
40
2O
o 100
o
o
15
80
60
40
20
60%
I
30%
M
10%
72%
21%
M
7%
59%
32%
M
9%
4
Day
B
68%
I
26%
M
6%
68%
I
24%
M
8%
77%
16%
M
7%
Day
Figure 35.
Interrelationship between the zoogloeal matrix and
absorption. (A) Addition of Zn65 at time of inoculation
of culture of Zoogloea ramigera 115. (B) Addition of
Zn^^ after 3, k, or 5 days incubation.
S = supernatant, M = matrix, C = cells
97
-------
e-
6
10
Time in Hours
Figure 36.
Uptake of Zn by a crude extract of exocellular polymer of
Zogloea ramigera 115. Zn^S plus 100 ng/ml carrier (H);
plus 10 ng/ml carrier (©).
-------
vo
TABLE 18. Values (mg/L) shewing difference in concentration of selected cations present
in pH 3.0 acid mine water before and after contact with cell-floe
Soluble Cation*
(oxidation state not determined)
Control (Acid mine water)
Sample (Avg. of U)
Change
Total removed from 50 ml
Millimoles removed
Al
180
120
-60
3mg
.11
Ca
360
285
-75
3.75mg
•09
Fe
^75
3^2
-133
6.65mg
.12
Mg
280
202
-78
3.9mg
.16
Mn
30
22
-8
0.1«ng
.007
Si
38
28
-10
0.5mg
.02
Soluble Anion
N03-
18.2
16
-2
O.lmg
.001
S04=
1*600
3^50
1150
57.5mg
.6
*Ag, Co, Cu, Cr, Li, Mo, Ni, Ti were all less than 2 mg/L in the mine water. They were determined
but not included in the table.
-------
detected was 1*8.1 millimolar and the total cation content was 38 millimolar.
By difference the undetected cation is 10 millimolar and is presumed to
be H"1" . This corresponds to a theoretical pH of 2 as compared to the
recorded pH 2.8 and probably reflects some undissociated HS04.
The total weight of ions removed by 300 mg cell-floe is calculated at
about 76 mg. This represents an accumulation of 25% of its own weight
by the cell- floe polymer.
It was assumed that the floe-polymer adsorbed cation and that the anion
was adsorbed by association with the cation. The reverse was also a
possibility and a series of experiments was carried out to examine the
influence of anions on uptake of cations.
Results of these experiments are summarized in Table 19. The amount of
Fe+3 as (FeCls) uptake by cell- floe was examined using two different con-
centrations of Fe (375 and 1J.60 mg/L. respectively, whereas the Cl~ ion
content was 750 and 4000 mg/L. respectively) . These analysis are desig-
nated as [A] in Table 19, and are compared to the uptake of FeCLg by
cells which had been previously washed with one of the following salts:
KH2K)4, KN03 or MgS04. Table 19 is divided into 3 sections: control or
stock ion concentrations, sample solution values after 18 hours in pres-
ence of floe, and calculated difference values giving the concentration
of ion removed (mg/L^ from a J50 ml flask.
Wet weight of cell- floe in these experiments ranged between 32.36 g and
37. 5^ g per 50 ml flask. The P0«», NOa and S0i» values in the sample
reflect the amount of POi*, NOa or S0«» taken up by cell-floes during the
washing procedure prior to addition of
Comparison of [A] values in Table 19 indicates that virtually all of the
iron was adsorbed by the floe when the initial concentration was 375 mg/L.
That is, from 3.7 to 7.0 mg Fe remained in solution. When the initial Fe
concentration was Il60 mg/L., 375 mg/1. remained after 18 hours indicat-
ing that the floe -polymer had reached its saturation point. However,
when the cells were washed with KH2P04 solution [B value] and then exposed
to 1160 mg Fe/L., all of the Fe was adsorbed. This suggests that P04 is
adsorbed in the cell-floe during washing and that Fe is associated with
the P04. The P04 values verify this observation. R)4 is taken up by floe
and during the process more Cl~ is also adsorbed by the floe. That is the
Cl~ difference is greater for the [B] values than for the [A] values.
The same general observations hold for the N03 and S04 columns. No con-
clusions are drawn as regards S04 because it was added as MgS04. Mg was
shown in Table 18 to be adsorbed.
Total weight of ions adsorbed to 300 mg dry weight floe in a 50 ml flask
is calculated as 270 mg in the case of 1160 mg Fe/L., and indicates that
the polymer has adsorbed nearly the equivalent of its own weight as FeCl3.
100
-------
TABLE 19. Values showing (A) concentration of Fe+++ and Cl" (FeCl3) absorbed by cell-floe,
and by cell-floe after washing with (B) any one of three different anions
Ion Concentration (mg/L)
Cell weight
wet
A 32.26
B 32.26
A 34.46
B 34.46
A 34.87
B 34.87
A 32.78
B 32.78
A 37.54
Control
Fe Cl
375 750
375 750
1160 4000
1160 4000
375 750
375 750
375 750
375 750
P04
900
900
-
-
N03 S04"
-
-
1320
480
375 750 -
*P04 added as KH2P04, NOs as
**Values not valid because of
Sampl e
Fe Cl
7 408
0.5 125
373 **
1.2 292
4.5 342
0.9 100
8.7 433
4.7 92
after 18 hr
P04 N03 S04
670
593
1065
390
3.7 370 - -
KN03, S04 as MgS04.7H20
analytical interference.
Fe
368
374
787
1158
370
374
3G4
370
370
Di
Cl
342
625
3708
408
650
317
658
380
fference
P04 N03 S04
230
307
255
90
_
-------
Since it was apparent that total Fe uptake by floe increased with increas-
ing concentration for a given time period, it was decided to examine this
phenomenon in acidic mine water. For this purpose adsorption of Fe from
mine water was compared to adsorption of Fe from mine water which con-
tained added FeCLg. Results of this experiment are tabulated in Table 20.
TABLE 20. Iron values (mg/L) showing adsorption of Fe from either pH 2.8
acid mine water or pH 2.8 mine water which was supplemented
with Fed
Weight of cell floe/flask
Fe control
Fe cell control
Fe sample (Avg. 3)
Fe difference
Total Fe adsorbed/50 ml flask
Total Fe adsorbed/g floe
Mine
340
370
0
146
224
11.
33
Water
mg
2 mg
mg
Supplemented
Mine Water
260 mg
600
0
412
188
9.4 mg
37 mg
Although the iron content was nearly doubled the amount adsorbed did not
increase. The weight of cell-floe was less in the supplemented samples.
However, when the weight of Fe adsorbed was calculated on a per gram of
floe basis, the uptake was nearly equal. Unfortunately, a complete ion
analysis for these samples was not made and differences in uptake of
other mine water ions may exist.
The adsorption of ions by a culture of microorganisms that produce extra-
cellular polymer has been demonstrated in Tables 18, 19 and 20. It is
also known that production of polymer by cells can be stimulated via
cultural manipulations. This would result in greater production of adsorp-
tion sites for ions if indeed the ions are adsorbed to the extracellular
polymer.
Table 21 presents data showing the uptake of Fe and other mine water ions
by polymer which was isolated and purified from the cultures previously
described. The data in Table 21 illustrate that mine water ions are
adsorbed by purified polymer. It must be pointed out that the purifica-
tion processes alter the physical properties of the polymer and it may
not be directly comparable to behavior in the culture situation.
Mine water cations are strongly adsorbed by the polysaccharide polymer
synthesized by Zoogloea ramigera UL5 cells. Association of anions with
the adsorbed cations is apparent. The relative adsorption, approximating
25$, for most ions, suggests a non-specific uptake. However, the relative
affinities of ions for the polymer is not obvious from the available data
and no conclusions are warranted with regard to selectivity.
102
-------
TABLE 21. Values (mg/L) showing uptake of mine water ions by 50 mg
purified polymer per 50 ml flask
Fe Ni Co Mn
Mine water 375 1.1 0.7 11.6
Polymer sample (Avg. k) 297 0.5 0.35 5-5
Difference (uptake) 78 0.6 0.35 6.1
Total uptake per 50 ml 3.9 mg .03 mg .017 mg 0.3 mg
Ion adsorption to polymer promotes flocculation and affords a means of
physically separating the ions in a highly concentrated form from mine
water. In spite of lack of cation specificity, significantly high
amounts of iron and sulfate ions were removed from solution to attract
consideration of the process for pollution abatement purposes in selected
situations. Such a process could become very attractive if ions having
a market value were present in the solution. Production of cell-floes
is a potentially low cost process and increased polymer synthesis by cells
is a possibility which could increase the ion adsorptive capacity of the
cultures .
Many chlorinated hydrocarbon insecticides have been isolated from surface
waters, usually in concentrations of less than 1 |ig/liter or 1 part per
billion (ppb). Significance of pesticide in water has been established
(25) . Our interest is in the fate of these chemicals in a water column
and particularly in their adsorption to silt and floe forming bacteria
which form contemporary sediment in lakes. Bacterial floe is an aggre-
gation of cells which results in a macroscopic bacterial clump that
settles from liquid leaving that medium less turbid. This type of growth
appears to result from physical, chemical, and biological interactions
when extracellular fibrillar polymers are synthesized by organisms
(13,
A preliminary study of aerobic bacteria isolated from Lake Erie indicated
that of 33 isolates tested in 6 different growth media, 19 formed floes
in at least one medium, whereas 10 formed floes in two or more of the
media. Two of the floe forming isolates were selected for further study
with regard to their ability to concentrate and accumulate the pesticide
aldrin from solution. One bacterium was an orange-red pigmented gram
negative (G-) rod, tentatively identified as either a Flavobacterium or
Protaminobact er . The other was a gram positive (G+) Bacillus species.
Our experimental procedure was as follows: The test organisms were grown
in shake culture at ambient temperature (22° ± 2°C) in nutrient broth
(8 g/L., Difco), harvested by centrifugation, washed 2x with distilled
water and resuspended in 25 ml of distilled water. Erlenmeyer flasks
containing 50 ml suspensions of bacterial floe were then placed on a
rotary shaker and 1 ml of aldrin dissolved in acetone was added to give
a final concentration of 1 x lO"6 g aldrin/ml or 1 part per million (ppm)
103
-------
After being shaken at 120 rpm for the desired time period, the flasks
were removed from the shaker and the floe was separated from the super-
natant "by centrifugation. The floes were washed twice with distilled
water and the washings were added to the original supernatant. The
pesticide exposure time was calculated as that period between addition
of aldrin to the solution and the separation of the second washing from
the bacterial floe. The floe and supernatant fractions were extracted
separately with a 3:1 mixture of heptane and acetone. The organic
solvent fractions containing the aldrin were concentrated by evaporation
and adjusted to a volume of U ml. Two (il of samples were injected into
an Aerograph model 200 gas chromatograph (GLC) utilizing an electron
capture detector.
Total aldrin adsorbed to bacterial floe vs. time is plotted in Fig. 37.
The theoretical maximum for adsorption calculated from a standard curve
was 1 ppm aldrin. Recovery values for aldrin varied with individual
experiments between 70 and 130$ (0.7 to 1.3 ppn)» possibly due either
to adsorption on glassware (7) j or to varying sensitivity of the electron
capture detector. Almost all of the aldrin adsorption to floe took
place within the first 20 minutes of contact. The amount of adsorption
in most cases remained nearly constant after 20 min. This was verified
statistically by single variable linear regression at both the 1 and 5%
levels of significance when aldrin adsorbed vs. time was compared. All
of the aldrin added to the G+ bacteria was recovered from the floe; none
was recovered from the supernatant. The recovery value averaged 88$.
The slope of the line is linear and f3 = 0 at both the 1 and 5$ signifi-
cance levels when floe weight vs. pesticide adsorbed was evaluated.
This is due to the high rate of aldrin adsorption within the minimum
time period required to obtain the first adsorption value (i.e. 12-15
min.). Lack of significant difference in adsorption by 0.0^1 g floe
compared to 0.087 g floe indicates that 0.041 g was sufficient to adsorb
all of the available aldrin.
Adsorption curves shown in Fig. 37 indicate a rapid uptake of aldrin for
the first 20 min until maximum theoretical adsorption is reached. As
aldrin is adsorbed, less is available in solution to be adsorbed. There-
fore, the floes effectively adsorb from more dilute solutions than antici-
pated by the initial test concentration. This would essentially represent
adsorption from lower more realistic pesticide concentrations found in
Lake Erie.
Data for the G~ organism show that the amount of aldrin adsorbed by an
equal weight of cell floes remained the same or increased only slightly
with time beyond 20 minutes and either decreased or remained unchanged
in the supernatant. The adsorption curve was linear at the 1 and 5$
significance levels, but only if the 0.0656 g. wt. was omitted from the
calculations. If included, the data was not linear at either level.
However, in neither case did p = 0, indicating a relationship between
adsorption and floe weight in this case. This results from a slower
adsorption by the G~ bacteria as compared to the G+.
10U
-------
H
O
6
5
4
3
'So 2
£ I
X
CM
•c 6
< 5
3
2
Theoretical Maximum
Theoretical Maximum
No Data
l
10
Figure 37.
30
50
0.041 g
0.087 g
0.0838 g
0.1020 g Ql066g
0.24IOg
QI900g
0.06 56 g
70 90 110
Time (minutes)
130
150
250
270
Curves showing adsorption of aldrin "by both (a) gram positive bacterial floe and
(b) gram negative bacterial floe vs time. Numerals with each curve indicate the
dry weight of bacterial floe used in each experiment. Broken or dotted lines
indicate extrapolation from the first experimental point to 0 time
-------
The concentrating effect of these bacteria is considerable. For example,
when 0.0^1 g of G+ floe adsorbed pesticide from 25 g water, it represented
a concentration factor of about 625 to 1 within 20 minutes. A similar
but slightly smaller amount of adsorption occurred with the G- organism.
Analogous findings have been reported for algae (57, 95).
Samples of natural sediment which were in the process of settling and
accumulating in Lake Erie were collected by specially designed sediment
collectors placed on reefs (^9). This sediment consisted primarily of
inorganic matter. In addition to being analyzed in a manner identical
to that described for bacterial floe, it was also examined on a Dohrmann
Instrument Co. model 200A microcoulometer.
Aldrin and dieldrin were detected in contemporary sediment by both GLC
and the microcoulometer. Additional aldrin added experimentally was
adsorbed and as shown in Fig. 38, after 10 and 70 minutes the concentra-
tion was almost equal. No aldrin was detected in the supernatant. The
data were linear at both the 1 and 5$ levels of significance and (3 = 0.
Contemporary lake sediments appear to accumulate pesticide from suspension
in a manner similar to that shown for bacterial floe. Floe-forming bac-
teria are common in the lake environment and experimentally have a rapid
and high adsorption capacity for aldrin. Organic "floe-like" bottom sedi-
ments from the eastern basin of Lake Erie have been reported (89) and
electron microscopic examination of contemporary sediments from Lake Erie
shows that it is a conglomerate of bacteria, diatoms, inorganic and
detrital particulates. Counts of 105 aerobic and 10s anaerobic hetero-
trophic bacteria have been obtained per gram wet weight of contemporary
lake sediment. In this regard, clay particles are known to adsorb pesti-
cide and lindane is known to adsorb to lake sediments (60). Pfister
et al. (77) reported that chlorinated hydrocarbons both behave as sus-
pended microparticulates and are associated with other microparticulates
including detritus in the water-column. Others (71) have established
that DDT was taken up from organic detritus by fiddler crabs. Significant
concentrations of chlorinated pesticides have been detected in algae and
lake bottom mud (55). It is known that actinomycetes, fungi and other
bacteria adsorb and concentrate pesticides from solution (20, 97) and that
microparticulates associate with microorganisms (76). Particulate organic
material has been considered a potentially important source of food for
filter feeding marine organisms (3) • The suggestion has been made that
most of the particulate organic carbon at depths shallower than 175 m in
the Atlantic Ocean off South America consisted of living organisms and
decomposable organic matter (52).
It is concluded that floe-forming microorganisms act as adsorbants for
other suspended microparticles including chlorinated hydrocarbons and
this represents a natural process for removal of microparticles from the
water-column.
Once settled from suspension the fate of pesticides is in question, but
they may be degraded under anaerobic conditions (51)• It is likely that
106
-------
10
9
_ 8
N
b
x 6
c
'\-
a 5
I 4
O
3
2
Initial quantity of Aldrin
Initial Plus Experimentally Added Aldrin
-0.25 g.
-0.27 g.
10
20
30 40 50
Time (minutes)
60
70
80
Figure 38. Curves showing amount of aldrin found in two different contemporary sediment
(silt) samples, and the additional aldrin adsorbed experimentally by the two
samples vs time
-------
pesticides concentrated in bottom sediments for even short periods of
time would exert an insecticidal effect on the "bottom insects and other
susceptible fauna. Jensen and Gaufin (5^) and Carlson (l8) have shown
that different species of stonefly and mayfly naiads have varying sus-
ceptibilities to the same pesticide. This may explain the disappearance
of certain insects from Lake Erie, such as mayflies, and the persistance
or increase of others. The same may hold true for other organisms in the
lake.
Electron microscopic examination of contemporary sediments from Lake Erie
shows that it is a conglomerate of bacteria, diatoms, inorganic and
detrital particulates. Figures 39& and 39b are photographs of a scanning
electron micrographs showing contemporary sediment. Diatoms and detrital
material are clearly visible.
Polymer Production
Zoogloea ramigera 115 in liquid, shaken culture becomes extremely viscous
and then semi-solid in the presence of 0.5 to 2.0 glucose, so that the
flask can be inverted without disturbing the culture. This is referred
to as total thickening or gelled culture. Figure hO illustrates the
extent of polymer synthesis when Z_. ra.im'gera was cultivated in a 250 ml
Erlenmeyer flask containing Crabtree's arginine - salts medium supple-
mented with 2$ glucose. A plug of culture has been removed to aid in
visualizing the consistancy of the material. Rate of total culture
thickening was observed to be dependent upon glucose concentration in
the culture medium. Cells cultured in basal medium which contained
fructose, galactose, glucose, lactose, mannose, soluble starch, or sucrose
as the supplemental carbon source, all produced abundant and nearly equiv-
alent amounts of polymer. Since the polymer synthesis increases in
response to the concentration of carbon source in the medium it would be
reasonable to assume that the organism would produce small amounts of
polymer in low organic environments. It could then be surmised that the
organism would be more prevalent in the aquatic habitat than previously
thought, but might not be recognized as Zoogloea because of lack of capsu-
lar material synthesis in the absence of high organic carbon nutrient
concentrations.
Paper chromatograms of acid-hydrolysed polymers isolated individually
from cultures grown in various carbohydrates, although revealing only
one carbohydrate spot when developed in isopropanol rwater or in butanol:
acetic acidcwater as reported by Friedman et al. (U2), revealed two
distinctly separate spots for glucose (Eg. 1.00) and galactose (Rg 0.8?)
when developed in an ethyl acetate;pyridine;water system. These corre-
spond with Rg values of known glucose and galactose in the same system.
It was concluded that the polymer was a polysaccharide composed of
glucose and galactose with glucose as the predominant sugar. Cells grown
in a basal medium plus fructose, galactose, glucose, lactose, mannose,
soluble starch, or sucrose all produced polymer consisting of only glucose
and galactose.
108
-------
Figure 39.
A and B are electron scanning micrographs of
contemporary sediment from Lake Erie Showing
diatoms, detritus and particulate matter
X5500
109
-------
Figure kQ. Photograph of a 50 ml culture of Z. ramigera 115 in a
250 ml Erlenmeyer flask laying on its side illustrating
the semi-solid consistency of the culture due to poly-
mer synthesis. A core plug has been removed from the
culture to aid in visualizing the material.
Since the carbohydrate source did not affect the composition of the extra-
cellular polymer, glucose was selected as the carbohydrate source for all
further investigations. The time sequence of polymer productions was
investigated in a 1^ L. fermentor apparatus. The cells were so firmly
enmeshed in the matrix polymer that it was not possible to obtain cell
counts. Therefore cell growth was followed by a total DNA determination
using a diphenylamine assay. Utilization of glucose available in the
medium was followed by a combination of the anthrone test for total carbo-
hydrate and Nelson's test for reducing sugar.
Figure ^1 represents the results of one study obtained from a 10 L. fer-
mentor culture over a period of ten days. Utilization of glucose avail-
able in the medium is related to viscosity of both supernatant and whole
culture aliquot and to accumulation of PHB. The level of glucose dropped
in 58 hours from 62 micromoles to k.O micromoles and was therefore not
available in the culture solution between 60 and 2hO hours when the vis-
cosity was observed to increase. PHB concentration rapidly increased
between 20 and ^O hours and reached a maximum between 60 and 70 hours
suggesting that PHB formation stopped when the glucose was spent. After
75 hours there was a steady decrease in the quantity of PHB. At approxi-
mately 60 hours, viscosity of both supernatant and whole culture showed
110
-------
o>
E
03
O
03.
o
CL
1.6
5c 1.3
1.0
0.6
0.3
WHOLE CULTURE
CULTURE SUPERNATANT
120 180
TIME (hr)
240
240
180
120
60
10
8
0
300
en
UJ
e
0 <2
UJ
o
c75
o
o
en
Figure Ul. Curves showing viscosity of both whole culture and
supernatant, sugar available in medium measured by
both Nelson's and anthrone tests, and PHB accumula-
tion in Z. ramigera 115 culture vs time. Growth in
10 L batch fermenter at 2k° ± 2°C
111
-------
a marked rate of increase suggesting a relationship between 1KB expendi-
ture and polymer synthesis. At 180 hours an accelerated rate of increase
in viscosity was observed. In the course of the study, the viscosity of
the supernatant increased from 1.00 to 9.5 centistokes. In the same time
period the viscosity of the whole culture aliquot increased from 1.0 to
680.0 centistokes. At this point, it became extremely difficult to centri-
fuge enough supernatant from the samples to allow viscosity measurement
of the supernatant.
The rapid disappearance of glucose from the medium as determined by both
anthrone and Nelson's tests could be due either to rapid metabolism of
glucose or to physical adsorption of the sugar by this organism. Z.
raml gera isolate 115 has been shown to adsorb and accumulate metal ions
and organic matter in its matrix and the glucose could be adsorbed to the
matrix polymer (Ul). Crabtree (1966, Hi.D. Dissertation, Madison, Wis-
consin) studying a different isolate (I-16-M), has previously associated
the rapid initial uptake of glucose with the accumulation of 1KB. In the
present study 1KB accumulated within the cell, then as the viscosity of
the supernatant and of the whole culture increased, the quantity of the
IHB decreased. Perhaps 1KB is an intermediate storage compound that is
utilized as a metabolite for extracellular polymer production or for
synthesis of active precursors.
Figure k2 represents the results of a similar study on another fermentor
culture over a period of twenty days. The DNA assay data approximates
the familiar bacterial growth curve. As the total amount of DNA reached
a nifl.yiTm.nn at 96 hours, over half of the available sugar had disappeared
from the medium. After 250 hours the reducing sugar present in the
medium leveled off at 0.08 micromoles/ml or less. The total carbohydrate,
as reflected by the anthrone test data, leveled off at 11 micromoles in
the same time.
In this study the viscosity of the culture supernatant increased from
1.0 to 10.3 centistokes. The viscosity of the whole culture aliquot,
which contained cells and polymer increased to 130 centistokes. In other
batch cultures the whole culture viscosity has been measured as high as
1055 centistokes before total thickening made viscosity measurement
physically impossible.
Figure ^3 illustrates the disappearance of starch from the growth medium
when it was added in place of sugar as the carbohydrate source. Curve A
shows that all starch was hydrolysed within 10 hours. Total carbohydrate
assayed by the Anthrone procedure decreased less rapidly than starch
indicating either that starch was hydrolyzed more rapidly than glucose
was metabolized or that glucose was converted to new polysaccharide. The
latter is a likely possibility because total Anthrone positive carbohy-
drate did not go below 20 micromoles per ml; whereas reference to Fig. U2
shows that Nelson's reducing sugar (in this case, glucose) was completely
utilized. One other explanation for rapid starch loss wjuld be production
of Scardinger dextrins (5 to 7 glucose units) that were not yet metabolized
but would not react with iodine as starch.
112
-------
(NELSON>S)
90 120 ISO ISO 210 240 270 300 330 360 390 420 450
Figure ^2. Curves showing viscosity of both whole culture and super-
natant, sugar available in medium measured by both
Nelson's and anthrone test, and total DNA in Z. ramigera
115 culture vs time. Growth study in 10 L batch fermenter
at 2^° ± 2°C
113
-------
STARCH HYDROLYSIS
(A)
ANTHRONE SUGAR
u
0.0
TlME(hr)
Figure ^3. Curves showing starch hydrolysis, change in viscos-
ity, and loss of total carbohydrate (Anthrone) in
Z. rsanigera isolate 115 culture vs time
Ilk
-------
The above observations have been made on 1? different fermentor runs
and the following interpretations seem reasonable. After the cells had
ceased to multiply as suggested by the leveling off of the DMA, vis-
cosity of both supernatant and whole culture continued to increase. This
continued increase in viscosity could be due to increasing molecular
weight, increasing branching, modification of the polymer such as a
change in water binding properties, or further synthesis of more short
chain polymer. Figure Ul shows PHB still decreasing after 180 hours.
The steadily increasing viscosity in the supernatants suggest the for-
mation of a polymer precursor or low molecular weight polymer which
remains in solution at 6600 x g. The molecular weight of the polymer
probably increases until it reverts from a soluble molecule to a sus-
pended molecule. Continued polymerization results in a matrix which
flocculates from suspension and ultimately forms a gel. Viscosity in
this situation can best be measured in whole culture aliquots to reflect
increase due to both suspended and dissolved polymer.
The efficiency of glucose and arginine conversion to polysaccharide has
been estimated from the data derived from several different fermentor
studies. Cellular DNA was assumed to be 3$ of the dry weight of the
cells. Comparison to DNA-growth curves indicated a production of U.6 g
cells/L. at the point of total culture thickening. This value was com-
pared to the total cell-floe dry weight value of 8.065 g/L which was
harvested from the culture vessel by centrifugation at the same time.
The calculations indicate that the cells comprise about 55$ of the total
cell-floe dry weight. Total ash values in these experiments accounted
for less than 6$ of the dry weight and it was assumed therefore that the
extracellular polymer accounted for about kQ%> of the weight. If it is
assumed that 50$ of the cell, dry weight and k&%> of the extracellular
polymer is carbon we can account for 3.83 g carbon/L of a theoretical
k.22 g carbon/L added as glucose plus arginine. The conversion of carbon
substrate to cells is therefore estimated to be 5^ and the conversion to
polymer is approximately 3*4$. It has been previously reported that the
floe-polymer adsorbs 99 x its own weight of water (32). That is, the
ratio of wet weight of culture gel to dry weight is 99:1'
Zoogloea ramlgera 115 has the capacity to produce prolific quantities
of extracellular polymer that has the capacity to adsorb metal ions and/
or organics from solution, to effect the removal of these materials from
water by flocculation and settling processes. Increased knowledge of
polymer production could lead to a workable system for industrial pol-
lution abatement.
115
-------
SECTION VI
GENERAL DISCUSSION OF THE IMPLICATIONS OF MICROBIAL POLYMER
SYNTHESIS IN WASTE TREATMENT AND LAKE EUTROPHICATION
The high, metabolic rates found in most bacteria establish them in a pre-
dominantly significant role in treatment processes.
Aerobic waste treatment plants are designed to take advantage of these
biological events. If the treatment system is viewed as a continuous
culture ecosystem comprised of a mixed microbial population, it can be
asserted in general terms that the biomass density is directly related
to nutrient concentration or sewage strength and the rate of BOD removal
(cell growth) is directly related to rate of flow through the treatment
plant.
As an ecological consideration there is no reason to consider the con-
version of oxidizable pollutants entering on oligotrophic lake or the
ocean (at the lowest trophic level) as anything different, except that
the overall system is more dilute and therefore the flocculent biomass
concentration will be less. In this regard, a highly eutrophic lake
such as Lake Erie, can be considered somewhere between an oligotrophic
lake and an aerobic treatment system. Indeed we find an intermediate
amount of flocculent sludge accumulation in Lake Erie. We are not so
crass as to imply that consideration of a lake system is not more compli-
cated than the above generalities may suggest - particularly at higher
trophic levels. However, at the lowest trophic level we can consider
the processes described as a by-pass around primary productivity which
contributes to the initial biomass formation both directly and by stimu-
lating primary productivity.
Relationship of Extracellular Polymer to Flocculation
We have been studying a group of bacteria that have been historically
recognized as primary contributors to aerobic waste treatment (1, 1^-, 15)
with particular interest in the organisms' ability to flocculate and to
concentrate substances from solution. Several floe-forming bacteria
have recently been isolated from waste water in our laboratory and else-
where by other investigators. The isolates from aerobic treatment systems
are characteristically gram negative pseudomonad type rods, some of which
produce an easily recognized zoogloeal or capsular matrix and are given
the generic name Zoogloea.
All of the floe-forming bacteria examined in this laboratory, with the
aid of electron microscopy, have been observed to produce extracellular
polymer fibrils. The polymer fibrils around the cells may be manifest
as a zoogloeal matrix, capsule, slime, or non-observable by bright field
microscopic techniques. Figure 27 shows the extracellular fibrils which
surround the cells that are characteristic of the type of flocculent
bacterium which appear as suspended particles but which do not produce
116
-------
the typical zoogloeal matrix. Figure 1? was prepared using a freeze
etching technique and shows the fibrillar strands that comprise the
zoogloeal matrix around Z_. ramigera 115. Several cells are embedded
within the fibrillar network of the matrix, and an interface that deline-
ates the matrix from the surrounding menstruum can be seen. Details con-
cerning matrix formation as well as fine structure and chemical compo-
sition of the extracellular polymer have been published elsewhere.
It has been postulated that the extracellular fibrils are responsible
for the flocculent growth habit of the organisms by entanglement of cells
among fibrils or by adsorption of cells to fibrils. The physical and
chemical properties of fibrils around different species of bacteria will
determine the extent to which water is bound to the polymer and will also
determine solubility properties of the polymer and affinity for adsorp-
tion of dissolved chemicals.
Chemical Adsorption by Extracellular Polymer
The zoogloeal matrix of isolate 115 as well as the floe of Z. ra.-migera
isolate I-16-M have been shown to possess a high affinity for transition
metal cations. For example, floes of Z. ram-igera 115 adsorbed the fol-
lowing ions from solution and increased the dry weight of the floe by the
percentage shown: $tfb of the weight as Cu+2, 25$ as Co+2, Y[% as Fe+3,
and 12.5$ as Ni+2.
The above data were obtained on floes in which cells were embedded. Sub-
sequent studies on purified matrix polymer isolated from Z. rajm-igera 115
established that the polymer behaves as a polyelectrolyte and also
adsorbs metal ions from solution. A collodial suspension of purified
polymer in the presence of certain metal ions will flocculate as a polymer-
metal ion complex and separate from solution or suspension. It appears
therefore, that the polymer fibrils which make up the characteristic
zoogloeal matrix are responsible for the unique adsorptive capacity of
the floe.
Relationship of ion adsorption to waste treatment and eutrophication. The
above observations can be used to explain some of the metal ion relation-
ships observed in activated sludge treatment systems. For example, the
schematic flow diagram of a municipal treatment plant shown in Fig. kk
shows the concentrations of some metal ions at various stages of the pro-
cess. The cation concentration of the supernatant after primary settle-
ing was generally less than that of raw sewage. The difference reflects
the amount of ion which was adsorbed by the settled sludge-floe. Since
adsorption from the bulk solution to floe represents a concentration from
a large volume to a small volume, the ion concentration in the anaerobic
digester was increased significantly. The concentration of ions in the
digester supernatant was 20 to 100 times greater than in the raw sewage.
This is recycled, giving a net buildup of ions in the mixed liquor when
compared to raw sewage. Figure ^5 represents data obtained at a later
date from the same system shown in Fig. kb, after the practice of return-
ing digester supernatant had been stopped. However, the data pertaining
117
-------
H
H
Co
RAW
SEWAGE
EOMGD
SCREEN
t
GRIT
CHAMBER _^ AE
t
t
*
*
.!
METAL ION
Cd
Cr
Cu
Fe
Mn
Ni
Zn
V
RETURN SLUDGE 5000 ppm ss
, ,
PRE PRIMARY
•RATON „ SETTUNG
: V
; SUPERNATANT
~~~
s
S
X
!
RAW\
SUJOCEv
~
AERATION
* / *
ANAEROBIC l>
DIGESTER MIXED
enrre LIOUOF
SLUDGE Y\
DISPOSAL H
u cl
CONCENTRATION OF ION IN /ig PER m* OF SUPERNATANT
0.
0.
0.
1.
0.
0.
0.
01
54
2
52
19
19
42
~
SETTUNG EFFLUENT
SUPERNATE ^
, 30MIN /
^P T Tl IMft 1 J
H I
FROM LOCATIONS INDICATED
0.01
0.
17 32.32 .6<
5
0.09 11.13 0.49
1
0.
0.
18
19
12 4.06 0.37
0.25 9.49 .8'
2
Figure hk. Flow diagram of a treatment plant (Canton, 0.) showing concentration
of metal ions at various locations
-------
H
H
RA
SEW;
SCRE
w
tGE
ZOMGD
£N
GRIT
CHAMBER '1*. AE
x
X
x
x
x
x
x
f
METAL ION
Cd
Cr
Cu
Fe
Mn
Ni
Zn
Pb
RETURN SLUDGE 5000 ppm ss
PRE PRIMARY
RATION fc SETTLING
0
*
*
f
*
t
' suuDGEv ANAEROBIC
; \ DIGESTER
t
'
I U
1
AERATION
x
X1
X1
X1
X
MDC
SLUDGE ^
DISPOSAL k
JJ
ID
OF
CONCENTRATION OF ION IN /*g PER m* OF SUPERNATANT
0.04
0.
3
0.04
1.06
0.22
0.28
0.48
0.28
0.
0.
0.
0.
0.
0.
02
3
06
55
20
21
0.30
0.
9
0.
FINAL
SETTLING EFFLUENT
X
X
SUPERNATE '
, 30MIN x
' SETTLING x
fl I
FROM LOCATIONS INDICATED
17 0.01
.74 0.07
0.86 0.02
9.75 0.2.2
.29 0. 2
0.99 0.23
2.86 0.05
0.79 0.21
Figure k5. Flow diagram of a treatment plant (Canton, 0.) showing concentration of metal
ions at various locations
-------
to mixed liquor and supernatant of settled mixed liquor indicates that
the metal ions were adsorbed to floe, and this sludge was recycled to
the system. The data presented in Figs. Ml and 1*5 are actual plant data
and are not experimental which unfortunately precludes a comparison of
values at each location throughout the system.
Organic adsorption to polymer and relationship to waste treatment and
eut roph icat ion. Laboratory investigations of the floe-polymer of Zi.
raml gera isolates 1 and 115 show that the floes actively metabolize" a
wide variety of aml.no acids, proteins, urea, and NH4+ as nitrogen sources.
Both isolates could hydrolyze casein, gelatin, starch and urea and
isolate 115 could hydrolyze collagen. The cells can use a variety of
organic acids, alcohols, sugars and sugar alcohols as carbon sources.
This suggests that proteinaceous and other natural organic wastes are
hydrolyzed by these floe-formers and any liberated amino acids would be
converted by amino acid oxidizing enzymes to corresponding organic acids
plus NH4+. The organic acids, liberated sugars, and KH4+ would then
serve to support growth and further extracellular polymer synthesis.
Polymer synthesis represents a BOD sink or removal mechanism when the
floes settle out. The effect is amplified because the polymers have
been shown to adsorb dissolved organics, such as amino acids, prior to
metabolism and would therefore remove more BOD than would be antici-
pated on the basis of metabolism. This latter effect is analogous to
biosorption described by Eckenfelder (see McKinney, 65) and can explain
the exceptional rate of BOD removal reported by Butterfield for these
organisms.
The available data is consistent with the following viewpoint: Settled
sludge of aerobic waste treatment processes is equivalent to the floc-
culent matrix described for laboratory cultures of microorganisms, which
are composed of cells, extracellular polymers fibrils and any detrital
or soluble compounds that adsorb to the fibrillar matrix. Under labora-
tory conditions the high molecular weight polymers adsorb large amounts
of water and monovalent (M+) cations (e.g. Na+, K4"). Polyvalent cations
can exchange with the bound water and M4", thereby releasing water and the
M*" ions. The polymer then becomes more hydrophobic and flocculation
results. The polymers are hydrolyzed under anaerobic conditions causing
a release of adsorbed or complexed metal ion back into solution, which
could be either recycled or discharged. Anaerobic digestion would
severely penalize the objective of mineral nutrient removal and could
contribute to algal growth and eutrophication if the discharge found its
way to receiving water. Conversely, the aerobic process is quite effec-
tive in removing metal ions and would benefit receiving water by with-
holding algal mineral nutrients. A controlled process could be exploited
as an advanced treatment procedure.
Flocculation in Natural Water
A large proportion of bacteria isolated from natural water are floc-
formers. That is, they grow in an aggregated manner forming massive
adherent particles (greater than 1.0 |im) rather than remain in suspension.
120
-------
For example, 19 of 36 bacteria isolated from Lake Erie formed floes in
the laboratory in at least one growth medium. Relatively few have been
examined using electron microscopic techniques to establish the presence
of extracellular fibrils. However, all of those which have been examined
in this manner do possess extracellular fibrils.
The interactions among floe-forming microorganisms, suspended and dis-
solved chemicals and other particulates (e.g. detritus and non-flocculent
microbes) are similar to those observed in waste treatment systems. A
greater variety of microorganisms will, however, be found in a lake
environment as compared to the ecologically more homogeneous treatment
systems. Some of these interactions have been described by Pfister et al.
It is worth emphasizing that the adsorption of chemicals by floe-formers
is a physical-chemical phenomenon related to chemical affinities and
interactions with toxic substances and inert substances as well as -with
nutrients are to be expected.
Figures ^46 through 50 illustrate some of the physical associations of
microorganisms and other particulate material that result in a conglom-
erate type floe which is the Lake equivalent of activated sludge. The
electron micrograph of Z. ramigera I-16-M cells (C) presented in Fig. k6
represents an experimental illustration of how insoluble suspended mag-
nesium silicate particles (P) can be adsorbed by extracellular polymer
fibrils (F). These associations increase floe density and increase the
ratio of inorganic to organic in sediment. Again, this results in a two-
fold adsorption effect because insoluble mineral particles are also known
to adsorb chemicals from solution. Figure ^-7 is a photomicrograph show-
ing individual bacterial cells (B) embedded in a capsular slime layer (S)
which surrounds a clump of Microcystis cells (M) taken from Lake Erie.
Figure U8 is a fluorescent photomicrograph taken at lower magnificat ion
than Figure 6 and shows the association of both bacterial floes (Bf) and
inorganic silt particles (P) with filaments of unidentified green algae
(GA.). Figure ^9 is a photograph of the identical field shown in Fig. k&
but taken under phase contrast illumination. The association of inorganic
particles with algae and bacterial clumps (fif) can be more easily seen
when Fig. 8 is compared to Fig. kQ.
Contemporary sediment which continuously forms and settles in Lake Erie
was collected by Herdendorf using specially designed sediment collectors.
This sediment is reported to have an average of about 10% organic and
90% inorganic content. Examination of the flocculent contemporary sedi-
ment shown in Fig. 50 after carbon replication and electron microscopy
clearly shows bacterial cells (B), a diatom (Di), fibrillar material (F),
and detrital material (De) which may either be organic or inorganic. As
previously discussed, microbial material can be exceptionally high in
mineral content and a 10% content of organics in sediments may be mis-
leading in terms of its microbial activity.
Both floe-forming bacteria and detrital particles have been observed to
adsorb chlorinated hydrocarbon pesticides in the laboratory and remove
121
-------
H
•V)
ro
B
Figure MS. Electron micrograph of a shadow
cast preparation of Z. ramigera I-16-M
with insoluble particles (P) of magnesium
silicate (talc) adsorbed to the extra-
cellular polymer fibrils (F) and cells
(C) X20,700
Figure U?. Photomicrograph of a wet mount
of blue green alga Microcystic cells (M)
containing bacteria (B) embedded within
the extracellular slime layer (S). Cells
were stained with dilute crystal violet
to increase constrast of bacteria XI300
-------
'„'
U)
Figure U8. Photomicrograph of an unstained
wet mount of naturally occurring algal-
bacterial floe to which a suspension of
insoluble fluorescent inorganic particles
had been added. The photograph was taken
under ultraviolet illumination and the
highly fluorescent inorganic particles (P)
can be seen in association with bacterial
floes (Bf) and green algal (GA) filaments
(see Fig. 1*9) X380
Figure ^9. Phase contrast photomicrograph of the
identical field shown in Fig. 48. The inorganic
particles can be seen by comparison to Fig. kQ
to be associated with flocculent bacterial
masses (Bf) in association with filamentous
green algae X500
-------
them from suspension. The contemporary sediment shown in Fig. 50 also
adsorbs chlorinated hydrocarbon pesticides and indeed contains a back-
ground content when obtained from Lake Erie. When the chemicals adsorb
to floe and settle from the water-column, it represents a natural puri-
fication process. When the sediment accumulates on the bottom, it repre-
sents a BOD zone and a relatively high localized concentration of certain
chemicals (e.g. pesticide). It is likely that the presence of high con-
centrations of pesticide in the sediment would exert an insecticidal
effect on bottom insects and other fauna.
On the basis of laboratory and environmental observations, the general-
ized environmental scheme outlined in Fig. 51 can be postulated. Dis-
solved organic and inorganic nutrients enter the water-column (Lake)
from a variety of sources including waste treatment effluents. Suspended
solids including both organic detritus and inorganic particulates also
enter from a variety of sources such as erosion and agricultural runoff.
Floe-producing bacteria and algae synthesize polymer from dissolved
organics which then adsorb more dissolved organics and inorganics, as
well as suspended organic and inorganics, and other living microorganisms.
When the resulting conglomerate reaches a critical mass and density
(related to chemical and physical parameters) it settles from the water-
column and accumulates on the bottom. This sediment is subject to either
wind and wave action whereupon it is resuspended and recognized as
increased water turbidity, or in deep zones it is subject to anaerobic
hydrolytic activity and soluble components are released into the water-
column to stimulate further ecological activity. Second order inter-
actions always take place and the ecosystem has been over simplified.
However, extracellular polymers produced by bacteria and algae probably
play a significant role in the natural purification of water and in the
eutrophication process.
12U
-------
Figure 50.
Electron micrograph of a carbon replica of contem-
porary sediment from Lake Erie
•.MICRO CARIIC jiAif
"*« «'l»
vfA; -
•&&-,-
X.'V '/".
I.JIW.HIN I. 'l'
^--^'
POlYMER
i Br
LGAE
SETTIEOFIOCCUIENT DETRITUS
ft ADSORBED CHEMICALS
DEEP ZONE BOTTOM SEDIMENT
Figure 51.
Schematic illustration of the interactions among
dissolved organic and inorganic pollutants with
suspended microparticles and polymers synthesized
by microorganisms
125
-------
SECTION VII
REFERENCES
1. Asai, T. 1968. Acetic acid bacteria, classification and biochem-
ical activities. University Park Press, Baltimore, Md., 3^3 pp.
2. Barer, R. and K. A. F. Ross. 1952. Refractometry of living cells.
J. Physiol. 118:38-39.
3- Baylor, E. R. and W. H. Sutcliffe. 1963. Limnol. Oceanogr. 8, 269,
k-. Sergey's Manual of Determinative Bacteriology. 1957- 7th Ed.
Breed, R. S., E. G. D. Murray, and N. R. Smith, eds. Williams
and Wilkins Co., Baltimore, Md.
5. Bernfeld, P. 1955. Amylases, a and p, p. 1^9-158. In Coldwick,
S. P. and N. 0. Kaplan (ed.) Methods of enzymology, Vol. 1
Academic Press, New York.
6. Bleiweiss, A. S., et al. 1967. Rapid separation of some common
intermediates of microbial metabolism by thin-layer chromatography.
Anal. Biochem. 20:335-338.
7- Bowman, M. C., F. Acree Jr., C. H. Schmidt, and M. Beroza. 1959-
Fate of DDT in Larvicide Suspensions, J. of Econ. Entomol. 52,
6:1038-10^2.
8. Bradley, G. and R. P. Williams. 1957- An electron microscope
study of the spores of some species of the genus Bacillus using
carbon replicas. J. Gen. Microbiol. 17:75-79*
9. Bachaman, R. E. 1917- Studies on the nomenclature and classifi-
cation of the bacteria. IV. Subgroups and genera of the coccaceae.
J. Bacteriol. 2:603-617.
10. Buck, T. C., and C. E. Reefer. 1959. Studies of a zoogloeal
forming organism found in activated sludge. Sewage and Indust.
Wastes. 31:1267-127^•
11. Burdon, K. L. and R. P. Williams. 1968. In Microbiology 2nd ed.,
McGraw-Hill Co., New York. p. ^38.
12. Burrows, W. 1968. In the Biology of the Microorganism. 19th ed.,
W. B. Saunders, Co., Philadelphia, Pa. p. 31-
13. Busch, P. L. andW. Stumm. 1968. Chemical interactions in the
aggregation of bacteria bioflocculation in waste treatment.
Environmental Science and Tech.
126
-------
2k. Butterfield, C. T. 1935. Studies of sewage purification. II.
A zoogloea- forming bacterium isolated from activated sludge. Public
Health Kept. U. S. 50:671-681.
15. Butterfield, C. T. 1937. The oxidation of sewage by bacteria in
pure culture. J. Bacteriol., 3^:207-219.
16. Butterfield, C. T. and E. Wattie, 19^1. Studies of sewage puri-
fication by trickling filters. Public Health Rept. U. S.
17. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf.
196U. Methods in Immunology, W. A. Benjamin, Inc., New York,
N. Y. 143 pp.
18. Carlson, C. A. 1966. Effects of Three Organophosphorus Insecti-
cides on Immature Hexagenia and Hydrosyche of the Upper Mississippi
River- Trans, of Am. Fish. Soc., 95, 1^:1-5.
19. Carr, J. G. 1968. Methods for identifying acetic acid bacteria.
In: Identification Methods for Microbiologists . Part B. B. M.
Gibbs and D. A. Shapton, Eds., Academic Press, New York, N. Y.
212 pp.
20. Chacko, C. I., J. L. Lockwood. 1967. Accumulation of DDT and
Dieldrin by Microorganisms. Can. J. of Micro., 13:1123-1126.
21. Cheronis, N. D., and J. B. Entrikin. 1963. Identification of
Organic Compounds. Interscience Publishers, New York, ^77 pp.
22. Cheronis, N. D., and T. S. Ma. 196U. Organic Functional Group
Analysis by Micro and Semimicro Methods. Interscience Publishers,
New York, 696 pp.
23. Clark, J. B. 1958. Slime as a possible factor in cell clumping
in Nocardia corallina. J. Bacteriol. 75:^00-^02.
2k. Clark, J. M. 196U. Experimental Biochemistry. W. H. Freeman
and Company, San Francisco.
25. Cope, 0. B. 1965. Research in Pesticides. Academic Press, New
York, pp. 115-127.
26. Crabtree, K., W. Boyle, E. McCoy and G. A. Rohlich. 1966. A
mechanism of floe formation by Zoogloea ramigera. J. Water Poll.
Control Fed. 38:1968-1980.
27. Crabtree, K., E. McCoy, W. C. Boyle and G. A. Rohlich. 1965.
Isolation, identification, and metabolic role of the sudanophilic
granules of Zoogloea ramigera. Appl. Microbiol. 13:218-226.
127
-------
28. Crabtree, K. and E. McCoy. 196?. Zoogloea ramigera Itzigsohn,
identification and description. International. J. Systematic
Bacteriol. 17:1-10.
29. DeToni, J. B. and V. Trevisan. 1889. Schizomycetaceae in
Saccardo's Sylloge Fungorum. 8:923-108?.
30. Dische, Z. 1955- Color reactions of nucleic acid components,
In E. Chargaff and J. N. Davidson (ed.), The nucleic acids, vol. 1.
Academic Press, Inc., New York. pp. 285-305.
31. Drake, C. M. 1965- Occurrence of Siderocapsa treubii in certain
waters of the Niedrrhein. Gewasser und Abwasser, Heft. 39/^0:
ia-63. ~^
32. Dugan, P. R. 1970. Removal of mine water ions by microbial
polymers. Proceed. 3rd Symposium on Coal Mine Drainage Research.
279-283. Mellon Inst. Pittsburgh, Pa. ho6 pp.
33- Dugan, P. R. and D. G. Lundgren. 1960. Isolation of the floe-
forming organism Zoogloea ramigera and its culture in complex and
synthetic media. Appl. Microbiol. 8:357-361.
3k. Dugan, P. R., C. B. MacMillan and R. M. Pfister. 1970. Aerobic
heterotrophic bacteria indigenous to pH 2.8 Acid Mine water:
Predominant slime producing bacteria in acid streamers. J.
Bacteriol. 101:982-988.
35. Dugan, P. R., R. M. Pfister and J. I. Frea. 1970. Implications
of microbial polymer synthesis in-waste treatment and lake
eutrophication. Preprints 5th Internat. Congress Water Pollution
III - 20/1 - 20/10. Pergamon Publ. Co.
36. Eyster, C. 196U. Micronutrient requirements for green plants,
especially algae. In Algae and Man. Plenum Press, New York, .N. Y.
37. Feigl, Fritz. I960. Spot tests in organic analysis. Elsevier
Publishing Co., New York.
38. Finstein, M. S. 1969. Growth and flocculation in a Zoogloea
culture. Appl. Microbiol. 15:962-963.
39. Fogg, G. E. 1965. Algal cultures and phytoplankton ecology.
Ch. 6. Univ. Wisconsin Press, Madison, Wise.
ij-0. Friedman, B. A. and P. R. Dugan. 1968. Identification of
Zoogloea species and the relationship to zoogloeal matrix and floe
formation. J. Bacteriol. 95:1903-1909-
128
-------
4l. Friedman, B. A. and P. R. Dugan. 1968. Concentration and accumu-
lation of metallic ions by the bacterium Zoogloea. Developments
in Industrial Microbiol. 9:381-388.
42. Friedman, B. A., P. R. Dugan, R. M. Pfister and C. C. Remsen.
1968. Fine structure and composition of the zoogloeal matrix
surrounding Zoogloea ramigera. J. Bacteriol. 96L2144-2153.
43. Friedman, B. A., P. R. Dugan, R. M. Pfister and C. C. Remsen.
1969. Structure of exocellular polymers and their relationship
to bacterial flocculation. J. Bacteriology, 98:1328-1334.
44. Ganapati, S. V., P. M. Amin and D. J. Parika. 196?. Studies on
Zoogloea colonies from stored raw sewage. Water and Sewage Works.
1157389^392.
45. Gibson and Colvin. 1968. Extension of bundles of cellulose
microfibrils on agar surfaces by Autobacter xylinum. Can. J.
Microbiol. 14:93-98.
46. Hardman, Y., and A. T. Henrici. 1939. Studies of freshwater
bacteria. V. The distribution of Siderocapsa treubii in some
lakes and streams. J. Bacteriol. 37:97-10$.
47- Harrington, B. J. and K. B. Raper- 1968. Use of a fluorescent
brightener to demonstrate cellulose in the cellular slime molds.
Appl. Microbiol. 16:106-113.
48. Henrici, A. T. and D. E. Johnson. 1935. Studies of freshwater
bacteria. II. Stalked bacteria, a new order of Schizomycetes.
J. Bacteriol. 30:61-93.
49. Herdendorf, C. E. 1968. Sedimentation studies in the South Shore
Reef Area of Western Lake Erie. Proc. llth Conference on Great
Lakes Research. 188-205.
50. Heukelekian, H. 194l. Mechanical f locculation and biof locculation
of sewage. Sewage Works. 13:506-520.
51. Hill, D. W. and P. L. McCarty. 1967. Anaerobic Degradation of
Selected Chlorinated Hydrocarbon Pesticides. J. Water Poll.
Control Fed. 32:1259-1277.
52. Hobson, L. A. and D. W. Menzel. 1969. The distribution and chemi-
cal composition of organic particulate matter in the sea and sediments
off the east coast of South America. Limnol. Oceanogr. lU_:159.
53. Horrocks, R. H. 19^9. Paper patition chromatography of reducing
sugars with benzidine as a spraying reagent. Nature. l6k:kkk.
54. Jensen, L. J. and A. R. Gaufin. 1966. Acute and long-term effects
of organic insecticides on two species of Stonefly Naids. J. Water
Poll. Control Fed. 38:1273-1286.
129
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55- Johnson, W. D., F. D. Fuller and L. E. Scarce. 1967. Pesticides
in the Green Bay Area. Proc. 10th Conference on Great Lakes
Research, pp. 363-37^.
56. Joyce, G. H. and P. R. Dugan. 1970. The role of floe-forming
bacteria in BOD removal from waste water. Develop, in Indust.
Microbiol. 11:377-386.
57- Keil, J. E. andL. E. Priester. 1969. DDT Uptake and Metabolism
by a Marine Diatom. Bull, of Environ. Cont. & Tox.,^, 3:169-173.
58. Law, J. H. and R. A. Slepecky. 1961. Assay of Poly-beta-hydroxy-
butyric acid. J. Bacteriol. 82:33-36.
59. Leshniowsky, W., P. R. Dugan, R. M. Pfister, J. I. Frea and C. I.
Randies. 1970. Aldrin: Removal from lake water by flocculent
bacteria. Science, 169:993-995.
60. Lotse, E. G., D. A. Graetz, 'G. Chesters, G. B. Lee and L. W.
Newland. 1968. Lindane Adsorption by Lake Sediments. Environ.
Science & Tech., 2:353-357.
6l. Luft, J. H. 1961. Improvements in epoxy resin embedding methods.
J. Biophys. Biochem. Cytol.
62. Marmur, J., 1961. A procedure for the isolation of deoxyribonucleic
acid from microorganisms. Jour. Molecular Biology. 3.:208-2l8.
63. Mclntosh, A. F. 1962. A serological examination of some acetic
acid bacteria. Antonie Van Leeuwenhoek. 28:^9-62.
6U. McKinney, R. E. 1956. Biological f locculation . In Biological
treatment of sewage and industrial wastes. Vol 1. 88, Rheinhold
Publ. Co., New York.
65. McKinney, R. E. 1962. Microbiology for Sanitary Engineers.
McGraw-Hill Co., New York, N. Y. 293 pp.
66. Moor, H. 196U. Die Gefrier -Fixation Lebender Zellen und ihre.
Anwendung in der Elektronenmikroskopie . Z. Zellforsch. 62:5^6-580.
67. Moor, H. and K. Muhlethaler- 1963. Fine structure in frozen-
etched yeast cells. J. Cell Biol. 17:609-628.
68. Morgan, G. B. 1961. The absorption of radioisotopes in certain
microorganisms. Quart. J. Florida Acad. Sci. 2U: 9^-100.
69. Muhlethaler, K. 19^9. The structure of bacterial cellulose.
Biochem. Biophys. Acta 3:527-535-
130
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70. Nelson, H. 19^. A photometric adaptation of the Somogyi method
for the determination of flucose. J. Biol. Chem. 153:375-380.
71. Odum, W. E., F. M. Woodwell, C. F. Wurster. 1969. DDT Residues
Adsorbed from Organic Detritus by Fiddler Crabs. Science, l6U : 576-
577.
72. Ohad, I., D. Danon and S. Hestrin. 1962. Synthesis of cellulose
by Acetobacter xylinum. V. Ultrastructure of polymer. J. Cell.
BiolT 12:31-46.
73 « Parsons, A. and P. R. Dugan. 1971. Production of extracellular
polysaccharide matrix by Zoogloea ramigera. Appl. Microbiol.
21:657-661.
7^. Pelczar, M. J. and R. D. Reid. 1965. p. 518. In Microbiology,
2nd Ed., McGraw-Hill Co., New York.
75- Peter, G. and K. Wuhrman. 1970. Contribution to the problem of
bioflocculation in the activated sludge process. Proceed. 5th
International Cong. Water Pollution. II - 1/1 - 1/9.
76. Pfister, R. M., P- R. Dugan and J. I. Frea. 1968. Particulate
fractions in water and the relationship to aquatic microflora.
Proc. llth Conf . on Great Lakes Research. 111-116. International
Assoc. for Great Lakes Research.
77- Pfister, R. M., P- R. Dugan and J. I. Frea. 1969. Microparticulates :
Isolation from Water and Identification of Associated Chlorinated
Pesticides. Science, 166:878-879.
78. Pfister, R. M. and D. Folger. 1968. Device for linear gradient
dehydration of specimens for electron microscopy. Appl. Microbiol.
79« Polikarpov, G. G. 1966. Radioecology of Aquatic Organisms.
Ch. 1. Rheinhold Publ. Corp., New York, N. Y.
80. Pribram, E. 1929. A contribution to the classification of micro-
organisms. J. Bacteriol. 28:361-39^.
81. Reddy, M. C., et al. 1969. Ester production by Pseudomonas fragi
II. Factors influencing ester levels in milk cultures. Appl.
Microbiol . 17 : 779-782 .
82. Rich, L. G. 1955. Respiration studies on the organic nitrogen
preferences of Zoogloea ramigera. Appl. Microbiol. 3:20-25.
83. Ries, H. E. and B. L. Meyers. 1968. Flocculation mechanism:
change neutralization and bridging. Science 160 :
131
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8k. Shilderkraut, D. 1962. Determination of the base composition of
deoxyribonucleic acid from its bouyant density in CsCl. Jour.
Molecular Biology. h_: 14.30-^3.
85. Skerman, V- D. B. 196?. A guide to identification of the genera
of bacteria. 2nd Ed., Williams and Wilkins Co., Baltimore, Md.,
303 PP.
86. Smith, I. 1958. Chromatographic techniques. Interscience
Publishers, Inc. New York.
87. Society of American Bacteriologists. 1957. Manual of Microbio-
logical Methods. McGraw-Hill Co., Inc., New York.
88. Steinecker, C. C. and M. S. Eheins. 1959. A micromodification of
the anthrone test for serum samples of limited quantity. Am. J.
Med. Technol. §5:377-380.
89. Storr, J. F. and C. J. Cazeau. 1969. Ecology of the Benthic
"Floe" in Lake Erie. Proc. 12th Conference on Great Lakes Research,
90. Tenney, M. W. and W. Stumm. 1965. Chemical flocculation of micro-
organisms in biological waste treatment. J. Water Poll. Control
Fed. 37:1370-1388.
91. Tezuka, Y. 1967. Magnesium ion as a factor governing bacterial
flocculation. Appl. Microbiol. 15:1256.
92. Tuttle, J., P. R. Dugan and C. I. Randies. 1969. Microbial
sulfate reduction and its potential utility as a water pollution
abatement procedure. Applied Microbiology. 17:297-302.
93. Uhz. R. F. and N. C. Dondero. 1967. The predominant bacteria in
natural zoogloeal colonies. I. Isolation and identification.
Can. J. Microbiol. 13:1671-1682.
9^. Unz, R. F. and N. C. Dondero. 1967- The predominant bacteria in
natural zoogloeal colonies. II. Physiology and nutrition. Can.
J. Microbiol. 13:1683-169^.
95. Vance, B. D. and W. Drummond. 19&9- Biological Concentration of
Pesticides by Algae. J. Am. Water Works Assoc., 61, 7:360-362.
96. Van Eeden, D. 1967. The antigens of Pseudomonas aeruginosa
studied by the Ouchterlony technique and immunoelectrophoresis.
J. Gen. Microbiol. WJ:95-105.
97. Voerman, S. and P. M. L. Tammes. 1969. Adsorption and Desorption
of Lindane and Dieldrin by Yeasts. Bull, of Eviron. Conta. &
Toxicology, k, 5:271-277.
132
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98. Wattle, E. 19^2. Cultural characteristics of zoogloe a- forming
bacteria isolated from activated sludge and trickling filters.
Public Health Rept. U. S. 57:1519-153^.
99« Wessman, G. E. and D. J. Miller. 1966. Biochemical and physical
changes in shaken suspensions of Pasteurella pestis . Appl. Micro-
biol. 1
100. Winslow, C. E. A. and A. F. Rogers. 1905- A revision of the
Coccaceae. Science 21: 669-672.
133
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SECTION VIII
LIST OF PUBLICATIONS
1. Friedman, B. A. and P. R. Dugan. 1968. Concentration and accumu-
lation of metallic ions by the "bacterium Zoogloea. Developments in
Indust. Microbiology. Vol. 9, 381-388.
2. Friedman, B. A. and P. R. Dugan. 1968. Identification of Zoogloea
species and the relationship to zoogloeal matrix and floe formation.
Jour. Bacteriol., 95:1903-1909.
3. Friedman, B. A., P. R. Dugan, R. M. Pfister and C. C. Remsen. 1968.
Fine structure and composition of the Zoogloeal matrix surrounding
Zoogloea ramigera. Jour. Bacteriol. 96:21^4-2153.
h. Friedman, B. A., P. R. Dugan, R. M. Pfister and C. C. Remsen. 1969.
Structure of exocellular polymers and their relationship to bacterial
flocculation. Jour. Bacteriol. 97:1328-133^.
5. Joyce, G. H. and P. R. Dugan. 1970. The role of floe-forming
bacteria in BOD reduction in waste water. Developments Indust.
Micro. 11:377-386.
6. Dugan, P. R. 1970. Adsorption of ions from mine water by micro-
bially produced polymers. Proceed 3rd Symposium on Coal Mine
Drainage Research. Mellon Inst., Pittsburgh, Pa. 279-283.
7. Leshniowsky, W., P. R. Dugan, R. M. Pfister, J. I. Frea and C. I.
Randies. 1970. Adlrin: removal from lake water by flocculent
bacteria. Science. 169:993-99^-
8. Dugan, P. R., R. M. Pfister and J. I. Frea. 1971. Implications
of microbial polymer synthesis in waste treatment and lake
eutrophication. Proceed. 5th Internat. Conf. Water Pollution.
(in press). Pergammon Press.
9. Leshniowsky, W., P. R. Dugan, R. M. Pfister, J. I. Frea and C. I.
Randies. 1971. Adsorption of chlorinated hydrocarbon pesticides
by microbial floe and lake sediment and its ecological implications.
Proc. 13th Conf. Great Lakes Res. 1970. 6ll-6l8.
10. Parsons, A. and P. R. Dugan. 1971. Production of Extracellular
Polysaccharide Matrix by Zoogloea ramigera. Applied Microbiol.
21:657-661.
131*
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PUBLISHED ABSTRACTS
1. Friedman, B. A. and P. R. Dugan. 1967- Accumulation of metallic
ions by zoogloea-forming bacteria, Abstracts S. I. M., p. 15.
2. Friedman, B. A., P. R. Dugan, R. M. Pfister and. C. C. Remsen. 1968.
Structure of the extra-cellular matrix which surrounds Zoogloea
rmrrigera floes. Bacteriol. Proceed., p. ho.
3. Friedman, B. A., P- R. Dugan, R. M. Pfister and C. C. Remsen. 1969.
Flocculation of bacteria by exocellular polymers. Bacteriol Proceed.,
p. 30.
U. Joyce, G. H. and P. R. Dugan. 1969. Ester synthesis by Zoogloea
rgmjgera 115. Bacteriol. Proceed., p. 28.
5. Joyce, G. H. and P- R. Dugan. 1969. The role of floe-forming
bacteria in BOD reduction in waste water. Abst. Soc. Indust. Micro.,
p. 18.
6. Leshniowsky, W., P- R. Dugan, R. M. Pfister and J. I. Frea. 1970.
Accumulation of chlorinated hydrocarbons by microbial floe and its
ecological implications. Abstr. 13th Confer, on Great Lakes Research,
Internat. Assoc. Gr. L. Res.
7. Parsons, A. and P. R. Dugan. 1970. Extracellular polymer synthesis
by Zoogloea ramigera. Bacteriol. Proceed. 70:58.
8. Schmidt, D., P. R. Dugan, F. W. Chorpenning and P. Griffith. 1970.
Antigenic relationships among the floe-forming Pseudomonadaceae.
Bacteriol. Proceed. 70:^0
9. Dugan, P. R., R. M. Pfister and J. I. Frea. 1970. Implications of
microbial polymer synthesis in waste treatment and lake eutrophica-
tion. Abstr. 5th Internat. Conf. on Water Pollution.
135
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DISSERTATIONS AND THESIS
Department of Microbiology
The Ohio State University
Ph.D. Dissertations
Friedman, B. A., 1968. Identification of Zoogloea species and its exo-
celliilar matrix, and the relationship of this matrix to floe-formation
and uptake of metallic ions.
Joyce, G. H., 1968. Biochemical studies of Zoogloea ramlgera isolate
115 with emphasis on ethyl alcohol metabolism.
M.S. Thesis
Parsons, A. B., 1970. Investigations of Zoogloea ramigera isolate 115
with emphasis on composition and synthesis of the extracellular matrix
polymer -
Leshniowsky, W. 0., 1970. Adsorption of the chlorinated hydrocarbon
pesticide aldrin by microbial floe and lake sediment and its ecological
implications.
136
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-75-032
2.
3. RECIPIENT'S ACCESSIOWNO.
4. TITLE AND SUBTITLE
BIOFLOCCULATION AND THE ACCUMULATION OF CHEMICALS BY
FLOC-FORMING ORGANISMS
5. REPORT DATE
September 1975 (issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Patrick R. Dugan
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Microbiology
Ohio State University
Columbus, Ohio U3210
10. PROGRAM ELEMENT NO.
1BBOU3
11. CONTRACT/GRANT NO.
17050 DFJ
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio U5268
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Several floe-forming bacteria vere isolated from polluted water by this and other
laboratories. All organisms studied produced extracellular polymer fibrils that
were related to slime matrix and flocculation. The extracellular polymers have
high adsorption capacity for: soluble metal and other mineral ions, soluble
organic nutrients (BOD), soluble toxic organics, insoluble mineral particles and
insoluble organic particulates. The bacteria remove BOD by physical adsorption as
well as by oxidative metabolism and can convert oxygen demanding organics to more
extracellular polymer. Production of polymer can be stimulated nutritionally to
yield amounts that have waste treatment - pollution abatement potential on a
commercial scale. The relationship of bioflocculation to waste treatment and lake
eutrophication is discussed and the basic mechanism of bioflocculation is considered.
Biochemical activities of individual floe-forming cells is examined because of its
relevance to polymer synthesis. Taxonomy of floe-formers is also considered in
relationship to biochemical activities.
17.
KEY WORDS AND DOCUMENT ANALYSIS
1. DESCRIPTORS
*Activated sludge process, *Adsorption,
*Aquatic microbiology—bacteria, *Colloids,
*Ions,*Radioactive waste processing—waste
disposal, *Sewage treatment—aerobic
bacteria, *Sludge, *Water pollution—sewage
treatment, Coagulation, Demineralizing,
Organic compounds, Pesticides, Slime,
Sedimentation. Radioactive wastes
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Syst emat i c s, Wast ewat er
treatment, Water purifi-
cation, Flocculating gels
13B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (Thispage}
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
137
ftUSGPO: 1975-657-695/5308 Region 5-11
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