WATER POLLUTION CONTROL RESEARCH SERIES
14010EGJ11 69
DAST-21
Effect of
Antibacterial Agents
on Mine Drainages
DEPARTMENT OF THE INTERIOR FEDERAL, WATER POLLUTION CONTROL ADMINISTRATION
-------
WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Reports describe
the results and progress in the control and abatement
of pollution of our Nations Waters. They provide a
central source of information on the research, develop-
ment and demonstration activities of the Federal Water
Pollution Control Administration, Department of the
Interior, through inhouse research and grants and
contracts with Federal, State, and local agencies,
research institutions, and industrial organizations.
Triplicate tearout abstract cards are placed inside
the back cover to facilitate information retrieval.
Space is provided on the card for the users accession
number and for additional keywords. The abstracts
utilize the WRSIC system.
Water Pollution Control Research Reports will be
distributed to requesters as supplies permit. Re
quests should be sent to the Publications Office.
Dept. of the Interior, Federal Water Pollution
Control Administration, Washington, D.C. 20242
-------
Effect of Antibacterial Agents on Mine Drainages
Use of Viable Antibacterial Agents to Reduce Pollution
by Mine Drainages
by
Robert E. Shearer
William A. Everson
FEDERAL WATER POLLUTION CONTROL ADMINISTRATION
DEPARTMENT OF THE INTERIOR
by
MSA Research Corporation
Evans City, Pennsylvania
for
COMMONWEALTH OF PENNSYLVANIA
DEPARTMENT OF MINES AND MINERAL INDUSTRIES
Grant
November 1969
-------
FWPCA Review Notice
This report has been reviewed by the Federal
Water Pollution Control Administration and
approved for publication. Approval does not
signify that the contents necessarily reflect
the views and policies of the Federal Water
Pollution ,Control Administration.
-------
TABLE OF CONTENTS
Section No. Page No .
1 Abstract iii
2 List of Figures v
3 List of Tables ix
4 Conclusions 1
5 Recommendations 3
6 Introduction 5
7 Construction Design of Experimental
Apparatus and Procedures 9
8 Isolation and Identification of Iron and
Sulfur Bacteria from Test Sites 11
9 Behavior of Natural Inhibitors 17
10 Field Tests in Robena Mine with Natural
Inhibitors 39
11 Attempts to Isolate, Concentrate and
Identify Phages 45
12 Caulobacters 57
13 Streptomyces as Producers of Antibiotic
Inhibitors 91
14 Acknowledgements 115
15 References 117
16 Glossary 119
Appendix No .
1 Statistical Analysis of Inhibition in
Various Streams 121
2 Analytical Data on Horizontal Flooded and
Vertical Spray Systems 123
-------
SECTION 1
ABS TRACT
A program was carried out to characterize the nature of
the active agents in certain natural waters that had previously
been found to inhibit bacterial production of acid in streams
through submerged piles of coal in plastic containers in the
laboratory. A further minor program was undertaken to test the
feasibility of application of the inhibitory principle to an
actual coal mine situation where acid was being produced.
In the mine study, two (2) inoculations of the natural
water previously found to be inhibitory were made in the amount
of about 14,000 gallons each to a workedout region of Robena
mine in Greene County, Pennsylvania. After the first inocu-
lation was made, laboratory tests showed absence of inhibitory
power in the water at the time it was collected; and the water
used for the second test was similarly found to be only weakly
inhibitory. No effect was noted in the drainage of the test
site over periods of several months after each inoculation. In
addition to the possibility of the weakness or absence of the
inhibitory principle, it is now believed that poor topography
of the floor of the mine of the test site might render appli-
cation of inhibitor ineffective. A more promising test site is
believed to be at Karen mine, also in southwestern Pennsylvania.
In efforts to characterize the nature of the inhibitor,
earlier, unreplicated, laboratory experiments were duplicated
on streams through submerged acid-producing piles of coal in
plastic containers. Inoculation of both raw and treated waters
previously found inhibitory resulted in a decrease in production
of acid, dissolved iron and sulfates and in an increase in pH.
Strains of Caulobacter which are bacteria characterized
by stalked appendages were found in the natural inhibitory
waters; and when adapted to acidic environments and concentrated
in culture media, they were inoculated into laboratory coal piles.
They then induced inhibition of the acid production. Other
adapted strains of Caulobacters obtained commercially also in-
duced inhibition. Some evidence was shown that Caulobacter in-
hibitors might move downstream in a flooded horizontal stream
through piles of coal refuse producing acid.
A group of sensitivity disks containing antibiotics made
by various species of Streptomyces was screened for effectiveness
against the ironsulfur bacteria producing acid in mines. The
four (4) species of Streptomyces producing the antibiotics found
to be effective were adapted to the acidic conditions found in
mine waters and were found effective against the acid-producing
bacteria in test tube cultures and on solid media.
111
-------
SECTION 2
LIST OF FIGURES
Fi ureNo, Page No .
1 Flooded Vertically Flowing System 18
la Flooded Vertically Flowing System-Detail 19
2 Effect of Inhibitor Upon Production of
Sulfate and Acid by Karen Bacteria 23
3 Ferrous and Total Iron by Karen Bacteria
with Filtrate Inhibitor 24
4 Ferrous and Total Iron Production by
Uninhibited Karen Bacteria 25
5 Ferrous and Total Iron Production by
Karen Bacteria with Raw Inhibitor 26
6 Effect of Inhibitor Upon pH in Karen
Cultures 27
7 Oxidation Times for Karen Bacteria 27
8 Inhibitors for Ferrous Iron Production
by Robena Bacteria 28
9 Effect of Inhibitor on Production of
Total Iron in Robena Cultures 29
10 Oxidation Times of Robena Bacteria 30
1]. Effect of Inhibitor on pH of Robena
Cultures 30
12 Effect of Inhibitor on Sulfate Production
by Robena Bacteria 31
13 Effects of Inhibitors on Acid Production
by Robena Bacteria 31
Flooded Horizontally Flowing System 33
14a Flooded Horizontally Flowing System-Detail 34
15 Vertical Spray Systems - Not Flooded 36
15a Vertical Spray Systems - Not Flooded-Detail 37
V
-------
LIST OF FIGURES (contd)
Figure No. Page No .
l6 Locations of Inhibitor Injection Poiflt :
and Drainage Pickup Points in Robena Mine
17 Zones of Inhibition on Agar L ayers and
Electron Micrograph of Caulobacters 60
18 Stock and McCormick Adapted Caulobacter
Cultures - Standard Agar Plates 62
19 Adapted Caulobacter Cultures Used to
Inoculate Coal 69
20 Two Electron Micrographs of Predominant
Bacteria in Effluents from Caulobacter -
Inhibited Systems 71
21 Effect of Caulobacter on Production
of Ferrous Iron by Robena Bacteria -
Test 2 76
22 Effect of Caulobacter on Production
of Ferrous Iron by Robena Bacteria
Test 1 76
23 Effect of Caulobacter on Production of
Total Iron by Robena Bacteria - Test 1 77
24 Effect of Caulobacter on Production of
Total Iron by Robena Bacteria - Test 2 77
25 Effect of Caulobacter on Sulfate Pro-
duction by Robena Bacteria - Test 1 78
26 Effect of Cau].obacter on Sulfate Pro-
duction by Robena Bacteria - Test 2 78
27 Effect of Caulobacter on Oxidation
Times of Robena Bacteria Test 1 79
28 Effect of Caulobacter on Oxidation
Times of Robena Bacteria Test 2 80
29 Effect of Caulobacter on Acid Pro-
duction by Robena Bacteria - Test 2 80
vi
-------
LIST OF FIGURES (contd)
Figure No. Page No .
30 Effect of Caulobacter on Acid Pro-
duction by Robena Bacteria - Test 1 81
31 Effect of Caulobacter in pH of Cultures
of Robena Bacteria Test 1 82
32 Effect of Caulobacter in pH of Cultures
of Robena Bacteria - Test 2 83
33 Effect of S. aureofaciens on Oxidation
Times of Robena Bacteria 113
34 Effect of S. aureofaciens on pH of
Robena Cultures in Upward-Flowing
Vertical Systems 114
35 Effect of S. aureofaciens on Acid
Production by Robena Bacteria on
Upward-Flowing Vertical Systems 114
vii
-------
SECTION 3
LIST OF TABLES
Table No, Page No .
1 Liquid Media Formulations (1) 12
2 Solid Medium - Thiosulfate Agar 12
3 Solid Medium - Iron Agar (1) 13
4 Bacterial Population of Test Site
Drainages 15
5 Additions of Inhibitor to Cultures of
Robena and Karen Bacteria 17
6 Viability of Karen Bacteria 21
7 Viability of Robena Bacteria 22
8 Dissolved Oxygen Content of Various
Streams 32
9 Dissolved Oxygen (ppm) at Various Ports
in Horizontal Systems 35
10 Addition of Inhibitors to Vertical
Spray Systems 38
11 Volumes of Trace Water and Inhibitor
Introduced Into Robena Mine 41
12 Analyses of Water From - 11 Right 42
13 Analyses of Water From - 12 Right 43
14 Drainage Sources as Filtrates Against
Iron Bacteria 49
15 Testing Adapted Caulobacters Against
Iron Bacteria 64
16 Steps in Attempts to Adapt Caulobacters 66
17 Viability of Robena Bacteria in vertical
Streams With and Without Caulobacter
Inhibitor 74
ix
-------
LIST OF TABLES (Contd)
Table No. e No .
18 Effects of Caulobacters Along
Horizontal Systems 88
19 Antibiotic Sensitivity Disk Testing 92
20 Testing Antibiotic Producer Products
Against Iron Bacteria 96
21 Inhibitory Effect of CoalGrown
Streptomyces (Sucrose Added) (16 days) 99
22 Inhibitory Effect of Coal-Grown
Streptomyces (Sucrose Added) (21 days) 101
23 Inhibitory Effect of CoalGrown
Streptomyces (No Additives) (17 Days) 104
24 Inhibitory Effect of Coal-Grown
Streptomyces (No Additives) (25 Days) 105
25 Inhibitory Effect of Coal-Grown
StreptomyceS (No Additives) (35 Days) 106
26 Analyses of Streptomyces Inhibited
Tubes 108
27 Testing for Presence of Streptomyces
Induced Iron Reduction 110
28 Viability of Robena Bacteria in Systems
Inoculated with S. aureofaciens 111
29 Viability of Robena Bacteria in Systems
Inoculated with S. spheroides 111
30 Test Data on Streptomyces as Inhibitor 112
31 Statistical Data on Characteristics of
Inhibited Streams 122
32 Total Acidity (ppm CaCO 3 equiv.) Along
Horizontal System 124
33 Total Iron Along Horizontal System 125
x
-------
LIST OF TABLES (Contd)
Table No. Page No .
34 Ferrous Iron Along Horizontal System 126
35 Oxidation Times of Bacteria From Along
Horizontal System (days) 127
36 pH Along Horizontal System 128
37 Sulfates Along Horizontal System (ppm) 129
38 Total Iron (ppm) Through Vertical Spray
System 130
39 Ferrous Iron (ppm) Through Vertical
Spray System 132
40 Sulfates (ppm) Through Vertical Spray
System 134
41 pH Through Vertical Spray System 136
42 Total Acidity Through Vertical Spray
System (ppm) 138
xi
-------
SECTION 4
CONCLUS IONS
Replicate tests confirmed presence of agents which in-
hibit production of ferrous sulfate from pyrites by bacteria
in an upwardflowing flooded vertical system observed in an
earlier project in unreplicated tests on McCormick mine effluent
and in waters from the region of Wildwood mine.
No proof was found of inhibition by unconcentrated
unoptimized natural inhibitor introduced into the bottom of
downward-flowing unflooded vertical systems, thus the inhibitor
could not be demonstrated to move vertically in an unsaturated
system.
No proof was found of inhibition by normal quantities
or copious quantities of unconcentrated unoptimized natural
inhibitor introduced into the influent of a flooded downward
flowing horizontal system.
A species of Caulobacter was isolated from the McCormick
mine effluent and when cultured in higher titer was found to
inhibit bacterial action on pyrites to form ferrous sulfate.
Other strains of Caulobacters obtained from the Ameri-
can Type Culture Collection were found to inhibit acid production
in flooded upward-flowing vertical systems. In an unreplicated
test, possible evidence of downward movement of inhibition by
Caulobacters on acidproducing bacteria was seen in a flooded
downwardflowing horizontal system.
An adapted strain of antibioticproducer (S. aureofaciens )
was found to inhibit acid production in replicate static tests
and in flooded upward-flowing vertical systems.
1
-------
SECTION 5
RECONNENDATIONS
Four (4) promising strains of Caulobacters should be
studied as inhibitors of acidproducing bacteria in upward-
flowing flooded vertical systems, which have been shown to be
susceptible to inhibition in the past. The most effective
strain from this and from two (2) strains already studied
should then be studied as inhibitors in flooded horizontal
streams and in unflooded downwardflowing vertical streams
when introduced into the bottom, since these systems seem to
be the most difficult to obtain inhibition.
One antibiotic-producing strain of Streptomyces
(S. aureofacien ) was adapted to acid mine conditions. This
eptomyces inhibited acid iron bacteria in replicate in
static tests and in upward-flowing vertical flooded systems.
This adapted microorganism should also be studied as an inhib-
itor under simulated mine conditions as given above. If
successful, its effects on aquatic life such as plants, fishes
and the like should be studied.
The most effective inhibitor should be inoculated into
a wide range of acidproducing regibns in mine workings, includ-
ing settling basins for fines from coal washings, caved-in under-
ground workings through boreholes and into high points in these
workings and over refuse piles with acid runoff.
3
-------
SECTION 6
INTRODUCTION
This is the final report on a project which was initiated
in July, 1968 and extended into the last half of 1969.
The obj ctives of this project were:
1. To extend laboratory results to
test the feasibility in coal mines
of reducing the production of acid
in drainages.
2. To characterize in the laboratory
the nature of the inhibitory mat-
erial.
The facilities of the Robena and Karen mines of United States
Steel Corporation were made available for this project. A truck-
load of coal from Robena mine was furnished for experiments,
samples of mine effluent containing acidproducing bacteria were
furnished from both mines, and a site where acid was being formed
was fitted out with plumbing to introduce inhibitors for field
tests. The background for this project and details of tests
are discussed in a later section.
MSA Research Corporation -had conceived the idea that
application of viable anti-bacterial agents to acid-producing
regions in coal mines might reduce the pollution in the effluents
of these regions. The Coal Research Board of the Commonwealth
of Pennsylvania supported two (2) programs in which a widespread
search for natural inhibitors was undertaken (Shearer, R.E. et
al 1968). Emphasis was first placed on bacteriophages, or
viruses which are specific for a given species of bacteria.
However, any viable agent antagonistic to acidproducing bacteria
wnich would proliferate in the mine waters would be suitable.
The search for inhibitors covered materials such as compost,
sewage, muds, neutral effluents from many mines and the like.
A new solid medium described in Section 8 had to be developed
for assay of the acid-Producing bacteria. Effective inhibitors
were found in the effluent of McCormickmine, Butler County,
Pennsylvania and in a region of Pine Creek, at the bottom of
a refuse pile at Wildwood mine, Allegheny County, Pennsylvania.
The effects of the inhibitors in lowering acidity and raising
pH were observed in effluents from flooded vertical beds of
coal in the laboratory as well as in static liquid and solid
culture. It was found that these natural inhibitors varied in
5
-------
effectiveness from time to time and that the nature of the in-
hibiting agent had to be established to insure dependability.
Some evidence was developed that one (1) of the inhibitory a-
gents might be a species of Caulobacter.
More recently, the present program supported jointly
by the Pennsylvania Coal Research Board and the Federal Water
Pollution Control Administration was undertaken to characterize
the nature of the inhibitor and to apply inhibitor to coal
mines that are producing acid. The first attempt to apply
natural inhibitor to a mine was unsuccessful. This may well
be due to the seasonal variability in the source of inhibitor
since a sample of this source drawn at the time the dose was
collected failed to show inhibition in the laboratory. The
nature of inhibitor had not then been established. Another
possibility is that the topography of the floor of the mine is
such that inhibitor did not flow into the acid-producing re-
gions. Another attempt was made with Wildwood water containing
the inhibitor after it had been found to have become effective
again. No inhibition was noted in the mine effluent in early
checks; but monitoring continues.
Replication of tests of natural inhibitor through
flooded vertical coal piles confirmed evidence of inhibition.
A study of movement of natural inhibitor showed no transfer of
natural inhibitor upward against a light stream flowing down-
ward through unsaturated coal piles nor horizontally through
a flooded horizontal bed of coal refuse. Horizontal movement
of a stock Caulobacter inhibitor was suggested in a test which
remained unreplicated because of depletion of funds.
The suggestion that a species of Caulobacter was inhib-
itory was partially confirmed in static laboratory tests and
in streams through vertical flooded beds of coal. Optical and
electron microscopy confirmed the presence of a species of
Caulobacter in the natural inhibitory waters of McCormick mine.
There remains to be determined the most effective species of
Caulobacter and its characteristics.
A battery of 15 antibiotics was tested against all species
of mine acid-proaucing bacteria and four (4) were found effective
against all of these. The various species of Streptomyces bac-
teria which produce these effective antibiotics under specialized
conditions were tested first on static laboratory cultures of
acidproducing bacteria and were found effective. Testing on
flooded vertical streams is still underway. The relative effec-
tiveness of these spec.ies and of the Caulobacter species under
various conditions needs to be determined to establish the optimum
inhibitor.
6
-------
With establishment of the optimum inhibitor, tests of
its effectiveness in actual mine conditions should be under-
taken. Sites are available for such tests.
7
-------
S E C I I 0 N 7
CONSTRUCTION DESIGN OF EXPERIMENTAL APPARATUS
AND PROCEDURES
Three different systems were designed to study
inhibition of acid producing bacteria in flowing streams in
coal piles. One was an upward-flowing vertical spray system,
one an unflooded downward-flowing vertical spray system,
and a downwardflowing flooded horizontal system. These
systems are described below. Inhibitors were tested either
as filtrates, as found naturally or in synthetic cultures.
Filtrates were prepared by introducing one volume of either
liquid cultures of iron bacteria from Karen and Robena mine
effluents into an equal volume of raw water from the Wildwood
mine. The bacteria were incubated for 24 hours, inhibitor
added, and the solution incubated an additional 24 hours, then
filtered through a 0.2 micron millipore filter.
Upward-Flowing Flooded Vertical Systems
Seven containers, each with a capacity of about 20
liters and measuring 18 1/2 x 11 1/4 x 11 1/4, were set up
to study inhibition in upwardflowing flooded vertical systems.
These containers were filled with sterilized run-of-mine coal
or coal refuse and with 9 liters of filtered tap water.
Chemical analyses showed the following composition: sulfur
2.2%, ash 10.5% and Fe 2 0 3 24.7%. Stainless steel tubes were
inserted into the coal so that filtered tap water could be
passed into the bottom of the pile. An outlet was arranged
so that an effluent would pass out near the top of the pile.
A photograph of the setup is shown in the section on experi-
mentation as Fig. 1. Alkali was first leached out of the coal
by passing dilute sulfuric acid through the piles. When
leaching was completed, as indicated by stabilization of p1-I
at a value of 5, acid-producing bacteria from Robena and Karen
mine effluents were inoculated into the systems and allowed to
propagate. When acidity developed, the test containers were
considered to be ready for inoculation of inhibitors.
Flooded Downward-Flowing Horizontal System
In practical application of inhibitors to mine drainage
systems, it might be desirable to introduce inhibitor to the
influent stream of an acidproducing region and then have the
agent move throughout the region. To determine if inhibition
might be obtained under these conditions, a set of troughs was
set up having an input of filtered tap water at one end and an
overflow at the other end. The troughs measure 10 feet long,
9
-------
1 1/2 feet wide and 8 inches deep were filled with sterilized
coal. Water (43 gallons) was added to a depth of 4 inches, the
height of the overflow. A photograph is shown in the experi-
mental section as Fig. 14. Alkali was leached out of the
coal with dilute sulfuric acid. The cultures of acidproducing
bacteria were added at the influent and allowed to propagate.
When acid was. developed, the systems were considered to be
ready for introduction of inhibitor.
Downward-Flowing Unflooded Vertical Systems
Some acid-producing regions in mines have water drip-
ping from the roof and walls. In these cases it would be de-
sirable to introduce the inhibitor into the water pool on the
floor of the mine and have the inhibitor move upward to the
source. Seven (7) plastic containers were fitted out to sim-
ulate this condition. These containers measure 42 inches high
and 15 inches in diameter and have sample ports installed 2 and
5 inches from the bottom and at intervals every 6 inches above.
the 5 inch point. The bottom port acted as the discharge point
for the acidic water. Spray nozzles were installed above the
top and adjusted to spray over the top of the coal at a rate
of 2 ml/min. A photograph of the setup is shown in the exper-
imental section as Fig. 15. Sterilized coal was loaded into
the containers. When the coal had been leached of alkali with
dilute sulfuric acid, cultures of acidproducing bacteria were
introduced and allowed to propagate. When an acid discharge
developed, the systems were considered to be ready for intro-
duction of inhibitor.
Design of Schedule for Chemical Ana yses of Various Systems
Standard methods were designed into a routine analytical
schedule for monitoring acidproducing systems. These include
the orthophenanthroline colorimetric method for dissolved fer-
rous iron. Total dissolved iron was also determined by this
method using hy4roxylaxnine to reduce ferric iron to ferrous be
tore adding the reagents. ( $tandard M thods for Examination of
Water and Waste Water , p-156 (1965) ).
The Sulfa-Ver turbidimetric method (Hach Chemical Co.)
was used for sulfates, and acidity was determined by titrating
hot solution with standard base to a pH of 8.3 using a pH meter
to determine endpoint.
10
-------
SECT I ON 8
ISOLATION AND IDENTIFICATION OF IRON AND SULFUR
BACTERIA FROM TEST SITES
Introduction
Since..inhibitors were to be used against the autotrophic
iron and sulfur bacteria from test sites at Robena and Karen
mines, it was first necessary to identify the species of iron
bacteria and to determine to what extent these autotrophic acid
producing bacteria were present at the test mine sites. Acid
drainages were collected and analyzed within a few hours after
collection to minimize any changes in the contents.
Identification Procedure
Aliquots of acid drainages from Robena and Karen mine
sites were inoculated into three (3) types of sterile liquid
synthetic iron media, and into thiosulfate and sulfur media to
identify the specific iron bacteria present. The compositions
of three (3) Fe II liquid media are given in Table 1. These
formulations are used as a partial test for identifying the
autotrophic iron and sulfur bacteria. The original inoculation
ratio was 1:10 drainage to medium. This was followed by a
secondary transfer of 1:100 after apparent bacterial growth was
evident as noted by ferrous iron oxidation, and/or oxidation
of thiosulfate and sulfur. The secondary transfer at higher
dilution was necessary so as to be certain bacterial, and not
chemical ,action was involved. After oxidation of media from
the secondary transfer, aliquots were plated on solid media,
using iron and thiosuif ate agars. The compositions of these
media are given in Tables 2 and 3. The composition for liquid
thiosulf ate is the same as given in Table 2 except that agar
was omitted. The pH was 4.5.
The formulation for the sulfur medium is given by
Churchill and Leathen, pl 8 (1961). The final pH was 4.0.
These media are used to biochemically identify the species of
iron or sulfur microorganisms present. Ten (10) neutral and
alkaline drainages were obtained from various mine areas within
the Robena mine complex and tested qualitatively for the presence
of iron and sulfur-oxidizing bacteria. It was found that iron
bacteria, and, in some cases sulfur bacteria were present, even
in drainages with a pH as high as 7.8.
Since these neutral or alkaline drainages contained
iron bacteria it appeared unlikely that they would act as in-
hibitors. However, they were tested as potential iron bacteria
inhibitors as described on page 47
11
-------
TABLE 1 - LIQUID MEDIA FORMULATIONS (1)
Compound Synthetic I Synthetic A Synthetic B
(NH 4 ) 2 S0 4 0.30 g 0.30 g 0.30 g
KC1 0.10 g 0.10 g 0.10 g
MgSO 4 6.72 g 1.00 g 1.00 g
K 2 HPO 4 0.10 g 0.10 g 0.10 g
Cad 2 1.94 g 0.22 g 0.22 g
1-tryptophane 0.02 g
(1) All media are made to 2 liters. The pH is
then adjusted to 3.9 with dilute H 2 S0 4 and 20
ml of 10% FeSO 4 solution are added to A and 13.
Fifty ml is added to I. The formulations are
our own modifications designed to maintain
good growth of iron bacteria and simulate
actual acid mine drainage.
TABLE 2 - SOLID MEDIUM - THIOSULFATE AGAR
Compound g/Liter Preparation
3.00 The base medium was
adjusted to pH 4.5
(NH 4 )S0 4 0.20 using H 2 S0 4 . The
medium was autoclaved.
CaC1 2 0.20 An aqueous solution of
10 per cent
NgSO 4 .711 2 0 0.10 Na 2 S 2 O 3 .5H 2 0 was
filter sterilized.
Ion agar 8.50 Fifty ml of the
(Na 2 S 2 O 3 .5H 2 0) was
Distilled Water 1000 ml. added to each 950 ml
of base medium, which
was poured into plates.
12
-------
TABLE 3 - SOLID MEDIUM - IRON AGAR (1 )
Component A
Agar Salts
Compound g/liter Preparation
(NH 4 ) 2 S 0 4 0.30 Adjust to pH 3.5
KC1 0.10 with H 2 5O 4 . Auto-
K 2 HPO 4 0.10 dave.
MgSO 4 2.00
Cad 2 0.22
Component B
Ferrous Iron
FeSO 4 7H 2 0 4.91 Dilute to 300 ml,
adjust to pH 3.5
with H 2 SO and
Morton Filter to
sterilize.
Component C
Agar
Ion agar No. 2 8.00 Make slurry in
150 ml H 2 0, heat,
then autoclave.
Mix 500 ml of A, 300 of B and 150 of C, then pour into
sterile Petri dishes. Plates last for 3 or 4 days.
(1) This formula is our own modification and supports
good growth of iron bacteria.
13
-------
Bacteria from the acid drainage of Robena area were
identified as the iron bacterium Ferrobacillus sulfooxidans
only, while both the iron bacterium,F. sulfo , and the sulfur
.iacterium Thiobacillus thiooxidans wire present in drainage
from Karen.
Three (3) types of liquid from media were used for iso-
lation and partial identification of iron oxidizing bacteria.
Drainages differed in the types of liquid iron medium oxidized
and the speed of oxidation. Synthetic I is a complex medium
simulating acid drainage to some extent. Some changes were
made in the salt concentration in hopes of providing a better
ionic environment for bacteriophage attachment to the iron
bacteria. A reference to this can be found in Adams, pp 141-144
(1959). Medium A does not contain as many elements and differs
from B only in that A contains a trace of 1-tryptophane, an
organic material. Bacteria isolated from cid drainage from
Robena mine area oxidized I and B, then A slowly. Iron bac-
teria are adapted to the environment within a particular mine.
Different mines, and even different areas within any one (1)
mine have differences in the environment. The iron bacteria
from various mines will oxidize iron more or less rapidly in
various synthetic iron media according to their ability to
adapt to the nutritional composition of each medium. This is.
the reason why ferrous iron media of various compositions
were used. An analogy can be made between isolating bacteria
from mines and soil in which no one (1) medium is adequate for
all species. This is pointed out by Alexander, p21 (1967).
L-tryptophane was added to medium A in order to deter
mine if iron bacteria could tolerate this organic material.
Initially the iron bacteria grew slowly in medium A, but sub-
sequent transfers showed that the rate of iron II oxidation was
increased as the iron bacteria adjusted to this medium. The
1tryptophane was also used as some bacteriophages require
this material in the medium to provide attachment to host bac-
terial cells. (Adams, pl45 (1959) and Bradley, p 2 58 (1967)).
Bacterial Population of Test Site Drainages
Total cell counts were made on the original acid drain-
ages from Karen and Robena. The results are shown in Table 4.
14
-------
TABLE 4 - BACTERIAL POPULATION OF TEST SITE DRAINAGES
Ident. No. Drainage Total Bacteria/mi
1 Karen - Discharge 2.5 x io
2 Karen - Fox Opening 3 x i0 7
3 Robena - Whitely 2 x io
4 Robena - Jenny Room 2.5 x io
These total bacteria counts may be high in comparison
with those given by other investigators. A thorough investigation
of the literature was not made, but other investigators used
different mine areas and waters for determining total cell counts.
There are undoubtedly many reasons for differences in
total cell counts. The total cell count is at best a semi-quan-
tative test and is of little significance since both living and
dead bacterial cells are counted. Changing conditions of water
flow, sampling sites, temperature, exposed coal surfaces, period
between sampling and counting, size and air space in collecting
containers, all influence the total numbers of bacteria.
These studies were not greatly concerned with determin-
ation of presence of bacteria other than iron and sulfur oxidizers
at the Robena and Karen test site. These drainages had pH values
averaging from 2.5 to 3.5. At these low pH levels very few
other types of bacteria can live. References to this can be
found throughout the literature, particularly in Alexander,
(1967), and Stanier, et al (1961). Thus the purpose of these
studies was to find bacterial inhibitors effective against the
iron and sulfur bacteria from the test sites and not to study
the complete flora of the waters.
One (1) test was conducted in order to determine if
Caulobacters were present in acid drainages from Robena and Karen
test site areas. Caulobacters were not found.
Bacterial Population of Wildwood Area
Total bacterial cell counts were not made on the Wild-
wood and McCorini.ck waters. The pH averaged about 7.0 when
samples were collected. Samples were collected along stream
soil banks indicating that high numbers of many kinds of micro-
organisms would be present at this pH. Culturing and identify-
ing all these microorganisms would be an impossible task since
conventional microbiological techniques can only estimate a
15
-------
portion of the total number of bacteria. A reference to this
can be found in Alexander, p-21 (1967).
The cell numbers are undoubtedly many times higher
than those in the acid drainages from Robena and Karen mines
since a count of only viable cells from the neutral mines
showed on the order of lO cells per ml. The viable counts
were made only on one type of medium (standard agar for
Caulobacters, Section 12). Use of other media more optimal
for enumeration of many types of soil and water bacteria
would give much higher viable cell counts, and thus still
higher total cells counts since both living and dead
bacterial cells are counted.
Chemical analyses were made on Wildwood water at
one time when the water was found to have a low pH. This
sample is not typical as most waters from this area were
neutral. The following values were obtained: pH = 4.5,
acidity = 37 ppm CaCO 3 equivalent, sulfate = 150 ppm, total
iron = 1 ppm, undissolved solids = 2800 ppm.
Chemical analyses were made at various times at
the Robena test sites and results given in Section 10.
16
-------
SECTION 9
BEHAVIOR OF NATURAL INHIBITORS
Unconcentrated raw waters from the Wildwood and
McCormick mines were tested against various acid-producing
strains of bacteria in streams through horizontal flooded
systems, in a vertical system with a light stream of tap water
flowing downward, and as a replicate of tests carried out in
earlier projects in streams through flooded vertical systems.
These were the waters that had shown inhibitor action in
earlier studies. Filtrates of these inhibitors were also test-
ed in similar streams. The various tests are discussed below.
Karen and Robena Mine Bacteria in Flooded Upward-Flowing
Vertical Systems
In order to replicate tests of inhibition of acid-
producing bacteria from Karen and Robena mines performed in an
earlier project, Karen bacteria were inoculated into three
upward-flowing vertical submerged coal pile systems (numbers
1-3), Robena into three others (numbers 4-6), and the seventh
remained as a sterile control. One of the systems is shown in
Figures 1 and la. When the bacterial populations were ob-
served by chemical and microbiological methods to be thriving
as indicated by viable cell counts and maintenance of low pH,
one of each of the piles seeded with Robena and Karen bacteria
was inoculated with the raw inhibiting water and one each
with the inhibitor filtrate. Reinoculations were also made
according to Table 5.
TABLE 5 - ADDITIONS OF INHIBITOR TO CULTURES OF
ROBENA AND KAREN BACTERIA
Type & .1 mount of Days After First Strain of
No. Inhibitor Addition Bacteria
2 100 ml raw 0 Karen
2 100 ml raw 27 Karen
3 100 ml filtrate 0 Karen
5 1000 ml raw 0 Robena
6 850 ml filtrate 0 Robena
6 100 ml filtrate 25 Robena
6 100 ml filtrate 52 Robena
17
-------
1
S
FIGURE 1 FLOODED VERTICALLY FLOWING SYSTEM
L
I
ii-
-------
COAL
WATER LEVEL
FIGURE Ia FLOODED VERTICALLY FLOWING SYSTEM.DETAIL
INFLUENT
19
-------
One of each pair of inhibitors was raw source as ob-
tained from the selected site at Wildwood mine. One of each
pair of inhibitors was similarly a filtrate obtained by intro-
ducing one volume of either Karen or Robena liquid bacteria
cultures into an equal volume of raw Wildwood drainage. The
bacteria were incubated for 24 hours, inhibitor added, and the
solution incubated an additional 24 hours, then filtered through
a 0.2 micron millipore filter.
The effects of the conditions are shown in Figures 2
through 13 and Tables 6 and 7. Oxidation times refer to the
time for liquid iron nutrient media to become cloudy. This
indicates bacteria activity compared to controls, which usually
takes 2 days to become cloudy. Viabilities are determined by
culturing bacteria from dilutions of effluent on solid plates
and counting the number of colonies. Inhibition of action of
acid bacteria and of oxidation times has been shown here. In the
case of viabilities, application of the statistical students
t test indicates statistically significant differences above
the 95% level between the controls and inhibitors on both the
Karen and the Robena streams as shown in the tables. Detailed
statistical analyses of the other variables of these tests
along with those of other tests are given in appendix 1
Inhibition in the fdooded upward-flowing vertical streams
is in contrast with the absence of inhibition in the vertical
spray and horizontal systems described below. In consultation
with representatives of the sponsors, it was decided to attempt
to determine the cause of the difference. One possible cause
was felt to be the difference in accessibility of air to the
streams since the surface areas of the containers testing Karen
and Robena were small relative to those of the other systems.
It was decided to aerate the containers for the 1 aren and
Robena tests to see if inhibition may be attenuated. Aeration
was initiated on the 116th day after first addition of inhibitor
to Robena piles and the 9th day after the addition to Karen
piles. Inhibition continued as before but the overall acidity
was at a higher level. Analyses were made of dissolved oxygen
content of the effluents made three days after aeration of con-
tainers 1 through 7 was started. Results are shown in Table
8 below.
20
-------
TABLE 6 - VIABILITY OF KAREN BACTERIA
Thousands of Cells per Milliliter
Days After #1 #2 #3
Inoculation No Inhibitor Raw Inhibitor Filtrate Inhibitor
0 2,4 3 0 28
8 l 4 0 5 008
13 309 1 2 7
20 14 6 O 8
28 7.6 4 2 0 6
34 8 6 404 1,8
44 7 l 304 101
51 13 l 0 0 5
57 705 14 0 0 3
64 3 4 405 0 3
71 7 2 0 6 0 3
79 1 6 001 0 2
85 1 7 001 0
92 1.5 0 4 001
99 2 0 12 4 6 4
107 l 6 0 4 6 3
113 2 5 007 5 2
120 1 7 1,5 0 7
127 1 0 0 7 1 4
134 0 8 18.8 705
142 10 3 2 0 2
150 4 2 <0 1 0 4
156 l 0 405 0 9
Statistical Analysis
#1 #2* #3
Sample Size 23 20 22
Mean 4.596 2.49 2.273
Standard
Deviation 3.00 (1&2) 3.48 (1 & 3)
Students t 2.29 2.24
Confidence Level >97.5%
* With two anomalously high values eliminated.
21
-------
TABLE 7 - VIABILITY OF ROBENA BACTERIA
Thousands of Cells per Milliliter
Days After #4 #5 #6
Inoculation No Inhibitor Raw Inhibitor Filtrate Inhibitor
0 305 3 109
1 25 4.1 2 ,V
4 4 0,2 0 ,8
15 3 1,7 0,2
22 3,9 2,0 1,5
30 1 0,8 0 ,2
35 1,8 0,4 0,5
42 3 1,7 0,6
40 2.8 0,6 0.3
56 3,6 0,9 004
66 4,0 0,3 0 ,2
73 3.5 0,8 0,3
79 1,8 0.2 0,].
86 3 ,2 1,1 0 ,6
93 1,7 0.4 0 ,2
101 1.2 0,1 0
107 0,3 0 0
114 0,4 0.2 <0,].
121 2.0 0.2 001
.129 1,1 <0,1 <0,1
135 0 ,1 0,2 001
142 1,2 0,1 0,2
149 0,5 0,1 11,5
156 0,8 <0 l <0,1
164 0,8 0,2 0,2
172 0,8 0.7
-------
I I I I I I I 1 I
Figure 2 - Effect of Inhibitor Upon Production of
Sulfate and Acid by Karen Bacteria
243C
2330
2200
1 00
1 100
1700
I4
I-I
0
I )
U
0
C)
Days After Adding Inhibitor
Days After Adding Inhibitor
-------
[ 1hf ihI Jni !T
70 80 90 100 110 120 130 140 150 160
Days After Adding Inhibitor
I I I I I I
I )
0 .
VEt
/
/
/
/
R
I
I
0 200
Figure 3 Ferrous ( ) and Total ( ) Iron by Karen Bacteria
with Filtrate Inhibitor
-------
120 1._ I I I I I I I 1 I I I I I _il
7lOI. T
Y
H
320
310 E
*10
10
150
140
130
120
110
100
4)
*0
70
40
50
40
30
20
10
I I
0
0
Si
0
0
0
80 90 100 110 12 (1 130 140 150 160 170
Days After Adding Inhibitor
Figure 4 Ferrous (_ . ) and Total C-) Iron Production by
Uninhibited Karen Bacteria
200 210 22(1
-------
J 1 T J I I I I I I i j i 1 1 1
304
300
111
I, I
I I
I
III
I I
0 50 20 30 II 30 40 70 so 00 100 114 130 130 140 iso i o 170 iso i o 200 210 220 230 210 250
Days After Adding Inhibitor
Figure - Ferrous ( ) and Total Iron (---) Production by
Karen Bacteria with Raw Inhibitor
3
a
SI
So
7
S
72
0
/ 1
26
-------
83 !iltrat./
- D c
5.0
4.0
3.0
Control \
p___ U
0 10 20 30 40 50 0 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210
Days After Adding Inhibitor
Figure () Effect of Inhibitor Upon pH in Karen Cultures
jI
0
U
0
4
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
Days After Adding Inhibitor
Figure 7 - Oxidation Times for Karen Bacteria
-------
0
- Ti hIhIhIIhIhIht
41hIhIhj!1T
650
40
A
430
420
6i0
340
370 E
210
200 E
140
170 E11 I
I
T I I
I I 4
:
If
i
I
I
I
I
I
ii
?
S I
I I
:
liii
Ii
t 1
:1
;II
I
I
o,
i
i1 ii
T
I
:
t I
0 10 20 30 40 00 40 70 40 90 100 110 120 t30 140 150 t o 170180 190 200 210 220 230 240
Days i fter ) dding Inhibitor
Figure 8 - Inhibitors for Ferrous Iron Production by Robena Bacteria
140
130
40
so
70
I 1
iio
t
0.
SO Tiltrats
28
-------
1 1 1 II I I [ J J I I I I I 1 J I I
741
730 E
f I
11
00$
330
330
310
300
390
200
234I TO STPΌ V O
E
11
ii l\r
Days After Adding Inhibitor
Figure 9 - Effect of Inhibitor on Production of Total
Robena Cultures
29
Iron in
I 1
11
7
F
110
100
90
I0
70
40
so
iv
Fi ltrst.
I
0 10 70 30 10 30
o
100 200 210
220 230 240 250 260
-------
18
18
14
12
10
4
2
0 10 20 30 40 50 80 70 80 90 100 110 120 130 140 ISO 180 170 180 190 200 210 220
Days After Adding Inhibitor
Figure 10 - Oxidation Times of Robena Bacteria
0 10 20 30 40 50 40 70 $0 0 100 110 120 130 140 150 180 170 180 190 200 210 220 230
Days After Adding Inhibitor
Figure 11 - Effect of Inhibitor on pH of Robena Cultures
30
-------
H
Figure 12 - Effect of Inhibitor on Sulfate Production by
Robena Bacteria
Days After Adding Inhibitor
Figure 13 Effects of Inhibitors
on Acid Production by Robena
Bacteria
H
STI W.TEt
I
H cc.txul
0 10 20 30 40 20 00 70 10 0 100 110 t20 130 140 150 t60 170 100 1 Q 250 210 220 235 240 234 200
Days After Adding Inhibitor
0 20 40 00 00 100 120 140 XI S 100 200
-------
TABLE 8 - DISSOLVED OXYGEN CONTENT OF VARIOUS STREAMS
Culture
No.
1
2
3
4
5
6
7
ppm
2.41
3.18
2.46
2.25
1.65
2.00
1.
78
Influent
water
1.94
There is wide variability in oxygen contents with no consis-
tent patters from which conclusions can be drawn except that
aeration is inefficient.
Horizontal System
In order to determine if unconcentrated natural
inhibitor might be effective when introduced in the influent
of flooded downward flowing horizontal systems cultures of
bacteria from an effluent from Robena mine were introduced
initially into three of the horizontal troughs. The experi-
mental setup is shown in Figure 14. A water flow was es
tablished at the rate of 15 mi/mm. When the bacterial pop-
ulation was established as determined by chemical and bacter-
ial analysis of the effluent a liter of raw natural mine
drainage supposedly containing the inhibitor was introduced
into trough #1 at the inlet. A second liter was introduced
after 34 days. Filtrate inhibitor (100 ml) was introduced
into trough 1 on the 37th day and another liter in the 63rd
day. To see if more abundant quantities of natural inhibitor
might be effective along the horizontal system, three liters of
inhibitor were added to one of the troughs. Results are given
in Appendix 2 . Here the first identifying digit represents
the trough number and the second the sample port number, start-
ing with the influent end. Thus 1-2 represents sample port #2
in trough #1. Some data points are lacking because mechanical
difficulties prevented sampling troughs. No inhibition was
noted from the unconcentrated unoptimized raw mine drainage
downstream along the horizontal system.
Dissolved oxygen was determined at various sample
ports along the horizontal systems on the 106th day after
first addition of inhibitor to one of them. Results are shown
in Table 9.
32
-------
I
f
T)
Ii
A
1
I
FIGURE 14
FLOODED HORIZONTALLY FLOWING SYSTEMS
-------
INFLUENT
SAMPLE PORTS
FIGURE 14a FLOODED HORIZONTALLY FLOWING SYSTEM.DETAIL
COAL
EFFLUENT
34
-------
TABLE 9 - DISSOLVED OXYGEN (ppm) AT VARIOUS PORTS
IN HORIZONTAL SYSTEMS
Sample
Port Numbers
1
2
4
5
9_
Trough
1
(with
inhibitor)
1.76
1.41
3.68
4.32
8.89
Trough
2
(no inhibitor)
1.59
1.71
2.92
2.86
6.48
The dissolved oxygen contents of these systems were significantly
greater at the effluent end than those of the laboratory coal
piles used to study inhibition of Karen and Robena bacteria. At
the influent end va2 ues are close to that of the tap water (about
1.9 ppm). It would pear that at the upper end of the pile
the oxygen is being consumed in the pyrite oxidation or the stream
is being aerated.
Unflooded Downward-Flowing Vertical System
Acid is often produced in. mines where water percolation
drips from the roof into pools in the floor. In order to de-
te xitine if the natural inhibitory waters studied in other systems
could be effective when introduced into pools at the bottom,
the unflooded downward-flowing vertical systems described in
Section 7 were put into operation. Cultures of bacteria were
added as follows: Ferrobacillus sulfooxidans to columns #1
and #2, Ferrobacillus ferrooxidans to #3 and #4, a mixture
of the two species to #5 and #6; and #7 remained sterile. The
setup is shown in Figure 15. After the bacteria had been well
established as evidenced by chemical and bacterial analyses
showing increased acidity and high viabilities in effluents,
inhibitors were added to the pool at the bottom of the column
in accordance with Table 10. Coal piles without added inhib-
itors were maintained as controls.
35
-------
It
1
I
-e
I
I
I
.
jI
! η
r u
FIGURE 15
VERTICAL SPRAY SYSTEMS - NOT FLOODED
I
- r
I
S-c
-------
SAMPLE
PORTS
1
FiGURE 15a
EFFLUENT
VERTICAL SPRAY SYSTEMS.NOT FLOODEDoDETAIL
SPRAY
NOZZLE
COAL LEVEL
WATER LEVEL
37
-------
TABLE 10 - ADDITION CF INHIBITORS TO VERTICAL SPRAY SYSTEMS
Days
After
Amount & Type First
Cultures of Inhibitor Addition
#2 F. Sulfo 300 ml raw Wildwood 0
F. Sulfo 800 ml Wildwood filtrate 48
#4 F. Ferro 300 ml raw McCormick 0
F. Ferro 800 ml McCormick Filtrate 48
#5 Mixed 150 ml each of 2 above raw 0
sources
400 ml each of 2 above
filtrates 48
No evidence of inhibition by unconcentrated unoptimized natural
inhibitors was shown in any of the cultures. Data are given
in tables in Appendix 2 . Throughout its first digit repre-
sents the number of the system and the second digit the number
of the sample port stating at the top (1-1, 2-1, 31, etc) and
ending with the pool at the bottom (1-7, 2-7, 3-7, etc).
Acidities were determined only on samples from the bottom port
because of the large size of the sample required for titration.
Data on tank #7 are not given after the 79th day because it was
flooded after that time. Some data, points are lacking because
mechanical difficulties prevented sampling. It was decided to
modify one of the containers to simulate the containers of
Robena and Karen cultures to see if inhibition would result.
This was done by sealing off the bottom sample port drain and
using the top sample port as the effluent. Filtered tap water
was introdtlced into the bottom. Total acidity fell from the
range of 200300 ppm to 20-30 ppm as a result of this action,
presumably because of lessened access of air to the system.
Other chemical properties are similar to those of the systems
for Karen and Robena bacteria.
38
-------
SECTION 10
FIELD TESTS IN ROBENA MINE WITH NATURP L INHIBITORS
The minedout area on the left side of 1 Main-lO Right,
United States Steel Companys Robena No. 2 Mine was chosen for
the test site. Mine water from this area flows naturally to
the two (2) pump pickup locations shown in Figure 16. The pH
of the mine water is normally about 3.0. The flow rate is
normally considerably less than 300 gpm, perhaps even less
than 100 gpm. The coal contours are such that water contain-
ing the inhibitor introduced in the abandoned 10 Right Flat
should flow thorugh the area to the 11 Right and 12 Right pump
pickups and, therefore, come in contact with the mine water
somewhere in the region.
Before attempting to introduce water containing in-
hibitor into the system, water from the mine fresh water system
was directed to the area with Calcocid Blue 2 G (Pylam Products
Company) dye to determine if it would report to the two (2)
pump pickup locations. Table 11 gives quantities introduced
and treatment of water used. 1he dye did not show up at the
pump pickups possibly because of adsorption by the gob; however,
an increase in the pH (presumably from dilution) from 2.7 to
as hLgh as 4.1 at the 11 Right and from 3.1 to as high as 4.2
at the 12 Right locations indicated that the water might have
traversed this area. See Tables 12 and 13.
Water from the region of Wildwood mine was injected on
February 24. It had a pH of 4.5 and contained 37 ppm total
acidity, 150 ppm sulfate, 2800 ppm unclissolved suspended sol-
ids and 1 ppm total iron. This was hauled to Robenas Hartley
Shaft in three (3) 4,500-gallon tank trucks from Wildwood Mine.
The 5inch fresh water line from Hartley Shaft bottom to 1 Main-
10 Right was separated from the fresh water system and connected
to a fire hydrant at the surface. The 13,500 gallons were
dumped into the hydrant and drained via the 5-inch line into
the 10 Right test area.
Two (2) samples were taken once each week at the 11
Right and 12 Right locations. Results are shown in Tables 12
and 13. No inhibition was noted possibly because topography
prevented access of inhibitor to some acid-producing regions
Furthermpre, because of variability in the natural inhibitor,
inhibitory power was absent at the time the water was collected
as evidenced later in laboratory tests, which showed no inhibition
in liquid cultures of iron bacteria. Laboratory tests made in
July 1969 showed that some weak inhibitory power had returned;
39
-------
\
LEGEND
A - Trace water and inhibitor
introduced at this loca-
tion.
B - 11 right pump pickup.
C - 12 right pump pickup.
\ fr-\
\.\
I \
\ 0
FIGURE 16 - LOCATIONS OF INHIBITOR INJECTION POINT
AND DRAINAGE PICKUP POINTS IN ROBENA MINE
N
\
N
N
N
N
N
\
\
\
FLOW
4
t
\\ 1\
40
-------
TABLE 11- VOLUMES OF TRACE WATER AND INHIBITOR
INTRODUCED INTO ROBENA MINE
Date
November 21, 1968
January 8, 1969
February 3, 1969
February 5,6,7, 1969
February 7, 1969
February 10, 1969
February 11, 1969
February 24, 1969
July 24, 1969
Gallons
12,000
13,500
12,000
102,000*
15,500
14,500
1,500
13,500
13,000
* This water added untreated to fill the local
depressions in the gob.
Dye
Added
Yes
Yes
Yes
No
Yes
Yes
Yes
Water from Wildwood
Mine Containing Inhib1t r
41
-------
TABLE 12- ANALYSES OF WATER FROM -
11 RIGHT
Date Total Total
Sampled pH Aciditj Iron
1/8/69 2.7 2720 493.0 5224
1/9/69 2.8 2720 504.0 5267
1/10/69 2.8 2840 526.0 4821
1/11/69 2.8 2910 538.0 4763
1/12/69 2.7 2880 543.0 4857
2/8/69 3.3 1800 375.0 4977
2/9/69 4.1 1000 336.0 4909
2/10/69 3.8 2150 717.0 4958
2/11/69 3.5 1600 428.0 4949
2/12/69 3.8 2750 818.0 4967
2/13/69 3.5 3550 582.0 5045
2/14/69 3.3 4250 459.0 4893
2/24/69* 3.0 3100 481.0 4767
3/3/69 4.4 3800 1075.0 5063
3/10/69 4.6 3550 620.0 3851
3/17.69 3.8 3400 810.0 4405
3/24/69 4.3 3500 750 4334
4/7/69 3.8 3530 870 4259
4/14/69 3.9 3600 710 4366
4/21/69 3.5 2950 560 4337
4/28/69 3.4 4700 1000 5315
7/3/69 3.5 2240 655
8/4/69 3.45 3230 ----
* -Inhibitor added. See Table Ii.
42
-------
TABLE 13 - ANALYSES OF WATER FROM - 12 RIGHT
Date Total Total
Sampled pH Acidity Iron S0 3 _
1/8/69 2.9 3110 1008.0 5228
1/9/69 3.1 3200 1002.0 4899
1/10/69 3.0 3180 1081.0 4809
1/11/69 3.1 3240 1008.0 4811
1/12/69 3.1 3400 1109.0 4755
2/8/69 3.0 3080 937.0 3939
2/9/69 3.3 3100 922.0 4230
2/10/69 3.2 3100 1002.0 4910
2/11/69 3.2 4200 1142.0 5009
2/12/69 3.5 4350 1165.0 4943
2/13/69 3.7 5400 1154.0 4990
2/14/69 3.4 5824 1165.0 4653
2/24/69* 4.2 5400 974.0 4586
3/3/69 3.4 6150 1198.0 4936
3/10/69 3.9 5550 820.0 4018
3/17/69 3.1 4800 955.0 4590
3/24/69 3.5 5250 925.0 4568
4/7/69 2.8 6200 1150.0 4322
4/14/69 2.9 4900 880.0 4847
4/21/69 2.9 5000 855.0 4384
4/28/69 3.4 5450 955.0 4791
7/3/69 2.9 3420 795.0
8/4/69 2.8 3390
* - Inhibitor added. See Table 11.
43
-------
and another field test at Robena was initiated in that month.
Progress will be monitored using company funds after completion
of the contract.
44
-------
SECTION 11
ATTEMPTS TO ISOLATE, CONCENTRATE AND IDENTIFY
PHAGES
Background
It seems in order to review some of the previous work
done on attempts to isolate phage specific for the iron bac-
teria and to review the criteria for proof of existence of
phage. Our previous work had.not given definite proof that
iron bacteria inhibition was caused by the existence of phage.
However, there were indications that some type of biological
agent was responsible for inhibition of iron bacteria. Previ-
ous experimental work can be found as given by Shearer and
Everson, (1967). Briefly, experimental results showed that
certain species of iron bacteria were lysed or inhibited by
Wildwood and McCormicK filtrates on agar plates. This In-
hibitory material could be transferred four or five times
without losing its potency. It is doubtful that a chemical
inhibiting agent could retain its potency through transfers,
particularly as each plate transferred extract was filtered
through a 0.20 micron filter. In addition, each filtrate was
specific for the strain of iron bacteria against which it was
prepared. Electron microscopy showed unidentified particles
associated with lysis of iron bacteria; however, no photographs
of a phage of known structure were obtained.
Prior to work on the present contract our proposal
st ed our attempts to identify phage had been unsuccessful,
but work on phage would be continued along with that on other
natural biological agents which might prove to be inhibitory
to iron bacteria. This includes competition from other species
of bacteria, specifically Caulobacter (previously found in
filtrates by electron microscopy). The Caulobacter may also
parasitize iron bacteria. Still other modes of inhibition may
be through the action of antibiotics, enzymes and bacteriocins.
Attempts to find phage (the only final proof being the demon-
stration o phage in electron micrographs) were continued in
our present study. Attempts were also made to determine if
viruses closely related to phage were present. A discussion
of this will be included in the following section on phage,
leaving work on Caulobacter and antibiotics as separate studies
included later on in this report.
Phage - Technical Methods of Study
The specific test iron bacteria were isolated from their
sources o acid mine water and grown in nutrient synthetic I
45
-------
acid mine drainage required for their maintenance and propagation.
Additional strains of autotrophic iron and sulfur bacteria were
obtained from the l merican Type Culture collection. The stock
strains obtained wete F. ferrooxidans , and F. sulfooxidans .
A search for phage required that potential phage
material be thsted against the 4 strains of iron bacteria,
the 2 stock cultures, £. sulfo isolated from Robena test site
and the F. sulfq isolated from Karen test site. It was necessary
to make requent transfers in order to keep these iron micro-
organisms viable.
Viral phages (bacteriophages) were sought in the natural
habitat of the host bacteria, i.e., soil, mud, mine drainage,
coal stockpiles and refuse dumps. The viable iron bacteria
were incubated in synthetic I mine drainage, then inoculated
with the potential phage source liquids in equal quantities.
After a second incubation period, the cultures were centrifuged
to partially remove bacteria and the supernates filtered through
a 0.20 micron millipore filter to remove bacteria. The filtrates
are tested for the presence of phage by:
a. Plating - Filtrate aliquots were diluted serially
by synthetic mine drainage and added in varying
ounts to each well-oxidized iron bacterium al-
ready growing in synthetic mine drainage. Aliquots
of this mixture were then added to soft agar and
the contents added to iron agar plates by pouring
over the surface of solid agar ahd rotating. After
solidifying, the iron agar plates were incubated
and the plates examined for plaques of lysis in the
film of iron bacteria groWth.
b. Lysis of liquid culture - The synthetic I iron
solutiolis were inoculated with actively growing
cultures of the various strains of iron bacteria
and filtrates. After incubation, the tubes were
examined for bacterial oxidation of ferrous iron
and compared with a bacterial control tube in-
oculated with iron bacteria but not with filtrate.
Phage or a lytic agent is indicated indirectly
when iron oxidation does not occur, or when tube
contents are partially oxidized. Suspected phages
were then further tested for by plating ΰliquots
from nonoxidized tubes as in (a). If phage was
obtained on an iron agar plate, it was picked or
eluted, millipore filtered, serially diluted and
tested against fresh iron bacteria on plates.
46
-------
Inhibition is often found on plates but not in liquids and the
reverse is also true. Adams, p-4 48 (1959) has observed that
a phage which brings about complete lysis of both cultures of
bacteria may produce very tiny plaques, may have a low effic-
iency of plating or a slow adsorption rate or may be other-
wise unsuitbale for quantitative work by piaque-copnting
techniques. Also, a phage which produces beautiful plaques
may not bring about lysis in broth cultures, may yield only
low titer stock or be difficult to work with in other ways.
The method of isolation may in part determine the properties
of the phage isolated, through isolation of variants.
Potential Phage Source Testing of Test Site Drainages
Ten neutral or alkaline drainages from the Robena and
Maple Creek complexes were prepared as bacteria-free filtrates
and tested on solid and liquid iron media. The filtrates were
prepared by inoculating each drainage into synthetic iron
cultures of each strain of bacteria, F. ferro (stock culture),
Karen mine iso1ated iron bacteria (F. sulfo) , and Robena
mineisolated iron bacteria (F. sulfo) . After incubation,
each drainage/bacterial culture was centrifuged and millipore
filtered to obtain the bacteriafree filtrate. This is one
method for detecting phage according to Adams, (1959). Each
filtrate was then tested against its specific strain of baη
teria on solid agar plates and in liquid synthetic iron media.
The tubes and plates were then incubated and observed for fer-
rous iron oxidation in liquid media and for appearance of
iron bacteria colonies on plates. Positive bacterial controls
were included in which bacteria were added to the tubes and
plates but no potential inhibitor. Tube and plate tests were
always compared with the positive bacterial controls. Negative
bacterial control tubes were also included in which neither
bacteria or filtrate were added to a tube of synthetic acid
mine drainage.
None of the ten neutral or alkaline drainages con-
tained an inhibitor against the iron bacteria.
Attempts to Isolate Phage from Other Mine Areas
Five potential phage sources from J & L mine were
also tested against the four strains of bacteria as previously
described. These sources were untreated drainages of neutral
pH. The prepared filtrates did not prevent ferrous iron oxi-
dation by each of the four bacterial strains in liquid media.
Slight bacterial reduction and plaques were seen on ilates con-
taining Karen bacteria and four J & L filtrates. It was evident
that four of the filtrates showed some slight inhibitory action
47
-------
on Karen bacteria. The five filtrates were not effective when
tested on solid media against the other three strains of bac-
teria.
A neutral drainage from the McCormick mine areas was
prepared as a filtrate and tested against the four strains
of bacteria. Ferrous iron oxidation was prevented in liquid
media for two days beyond the control culture of stock F. ferro .
Plaques, but no reduction in density of growthin non-plaque
areas, appeared on plates of F. ferro and filtrate, However,
results of testing the McCormick source against the other
three strains of iron bacteria were negative in that no bac-
terial inhibition occurred on solid or liquid media. This is
shown on Table 14.
Three Wildwood mine waters, neutral or slightly acidic,
were collected from various points within the mine area. These
water sources were also prepared as bacteria-free filtrates
against three strains of iron bacteria, F. sulfo , Karen and
Robena. Plates of iron bacteria isolated from the Karen and
Robena mines were partially clear of bacteria at 1:5 and 1:10
filtrate to bacteria ratios. The plates at a 1:100 and 1:1000
dilution ratios showed little clearing, but many plaquelike
areas in the growth of Karen iron bacteria. Plaque-like areas
were also seen at 1:100 dilution on the plates of Robena iron
bacteria. This occurred when filtrates from all three sites
were tested against their respective bacteria. The plates of
F. sulfo , while not as clear of bacteria as those of Robena
and Karen, still showed some clear areas and plaques. Filtrates
appeared to have a greater or lesser inhibiting effect on one
or another strain of bacteria, but differences were not great.
The filtrates were also tested against bacteria in
liquid synthetic iron media. It was observed that although fer-
rous iron was oxidized, the supernate liquid remained clear for
varying periods of time in some tubes. The bacterial control
tubes not only oxidized ferrous iron in two days, but also
became cloudy. It seems reasonable to conclude that tubes with
a clear supernate showed some evidence of partial bacterial in-
hibition. All results are shown on Table 14.
Three Wildwood filtrates partially inhibited Karen
iron bacteria for 10 days, while Robena iron bacteria were
partially inhibited for 5 days. No evidence of clearing was
seen in tubes of stock cultures of F. sulfo and filtrates. It
appeared that the phage or some other agent was inactivating
some of the iron bacteria. The remaining iron bacteria devel-
oped a resistance to the inactivating agent and started multi-
plying again to oxidize the iron,
48
-------
________ _____________ Test Iron Bacterium ______________________ _______________
Karen Mine - F. sulfo
Robena Mine - F. sulfo
Robena Mine - F. sulfo
Stock-Ferrobacillus
sulfooxidans (3)
Stock-Ferrobacillus
ferrooxidans (3)
Stock-Ferrobaci ilus
ferrooxidans (4)
Stock-Ferrobaci lius
ferrooxidans (4)
S tock-Ferrobaci 1 lus
sulfooxidans (3)
Karen Mine - F. Sulfo
Robena Mine - F. Sulfo
(1) Iron bacteria isolated from Karen and Robena mine sites were tested.
(2) Synthetic iron medium I as described on Table 1 was used for the iron oxidation tests.
This medium was used as it more closely resembles true acid drainage than A or B media.
(3) (4) These iron bacteria cultures were obtained from the American Type Culture Collection
and are referred to as stock.
TABLE 14
Waters
Waters
Waters
Waters
Source
of
Filtrate
Wildwood
Wi idwood
Wildwood
WI idwood
Samples
at
Various
1
1
2
1
- DRAINAGE SOURCES TESTED AS FILTRATES AGAINST IRON BACTERIA (1)
Solid Iron
Agar Medium
Taken Lowest
Filtrate: Bacterium
Times _________ ____ Ratio Showing Plagues
______ 1:1000
_____ 1:100
1:1000
Liquid Iron
Medium (2)
Inhibition of
Iron Oxidation
Days
10
5
0
Wildwood Waters
Wildwood Waters
McCormick Drainage
McCormick Drainage
McCormick Drainage
McCormick Drainage
2
2
1
1
1
1
1:100
0
No plaques at
1:1 dilution
0
No plaques at
1:1 dilution
0
1:100
2
No plaques at
1:1 dilution
0
No plaques at
1:1 dilution
0
No plaques at
-------
The Wildwood filtrates previously tested against
Karen and Robena bacteria and showing partial inhibition of
iron oxidation were retested in liquid mediat as before, the
ferrous iron was partially oxidized while the supernate re-
mained clear. Aliquots were plated on iron agar. Aliquots
from Karen and Robena control tubes were also plated at the
same time. In both cases, a viable colony count showed that
the filtrates reduced the bacterial counts in half.
Viable counts of 8 x lO and 6 x cells per ml
were obtained from the Robena and Karen iron bacterial controls
respectively, while the count from the Robena iron bacteria
with filtrate was 5 x 1O 3 cells per ml; the count from the
Karen iron bacteria and filtrate tube was 3 x 10 cells per ml.
Higher cell counts should have been obtained but
the iron agar plates were not freshly prepared and aliquots
were plated 6 days after inoculation of tubes. This is beyond
the peak growth for iron bacterial controls [ 2-3 days].
Fresh drainage sources were collected from Wildwood
and McCormick areas and two-liter quantities of filtrates
prepared against F. sulfo , F. ferro and Robena bacteria. This
was done in an effort to produce better inhibitors against
these bacteria. A three-liter quantity of effective inhibitor
was previously obtained against Karen iron bacteria. See Table
14, sample 1.
Filtrates were prepared and tested against stock
strains of F. sulfo and F. ferro . A filtrate was also tested
against Robena bacteria. Although none of the fresh filtrates
were effective in liquid media, a high degree of effective-
ness was demonstrated against Robena bacteria by a Wildwood
filtrate on solid agar plates. The filtrate inhibited most
of the Robena bacteria even when diluted as high as.l000
times, then tested against Robena bacteria at a 1:5 filtrate
to bacteria ratio. This data is also shown on Table 14.
However, none of the recent sources showed inhibition against
stock strains of F. sulfo and F. ferro on solid media.
A two-liter quantity of filtrate active against
Robena bacteria was stored in the deep freeze and was later
tested against Robena bacteria in flooded systems and troughs.
All Wildwood and McCormick waters were also tested
directly against iron bacteria. Small numbers of plaques
were sometimes obtained, but no inhibition of iron oxidation
in liquid media was seen.
50
-------
Various areas along the banks of a stream were
tested frequently at the Wildwood mine throughout the project.
Coal refuse lined the banks of the stream and the stream bed
was composed of fine coal silt. It was found that a natur-
ally occurring inhibitor appeared frequently during the late
spring and summer months of 1968 and 1969. During the winter
months, the inhibitor was not found. The pH was almost in-
variably somewhere near neutrality. Alexander, pp 26-27 (1967),
has stated that most microorganisms are mesophiles (bacteria
that grow best at 25°-35°C). The mesophilic types constitute
the bulk of the soil bacteria, but certain mesophilic species
develop best at temperatures below 20°C, These are cold-
tolerant species. Usually, the viable population is greatest
during the spring and autumn, A population burst occurs in the
spring as the soil becomes warm and organic materials become
accessible. The population commonly diminishes in winter and
remains in a state of biological inactivity, The seasonal
changes in numbers of bacteria are closely related to fluctua-
tions in moisture and temperature. Alterations in temperature
and moisture during the year may influence the bacteria direct-
ly. Climate factors may in part operate indirectly through
surface vegetation which is the source of carbonaceous-
nutrients reaching the microflora.
The McCormick area drainage was not tested as often
as Wildwood because it is a very small mine making it
difficult to obtain large volumes of water for inoculation.
Very recent testing of. Wildwood water was conducted
using serial dilutions of both drainage and filtrate. The
tests were conducted in parallel and plated on iron agar.
Tests results showed that both drainage and filtrate were
effective against Robena iron bacteria (F. sulfo ) at a ratio
of filtrate to iron bacterial culture of 1:100. Clear areas
of lysis were seen on the plates at 1:5 and 1:10 dilutions.
Large clear areas of iron bacterial lysis were not seen at
1:100 dilutions, but numerous small clear plaques were present.
At this time, July 1969, Wildwood waters were added to Robena
mine test site. Tank trucks carrying 13,500 gallons were
loaded with water from Pine Creek at the Wildwood mine. This
is in excess of the amounts spelled out inour proposals: 115
gallons for a flow of 20,000 gpd in the first draft and as
much as 10,000 gallons for a flow of(lOO gal/mm. The flow
from the gob region in Robena was not known with a high degree
of accuracy, but probably is less than 100 gpm or 144,000 gpd.
Dosage was thus one hundred thirty five times the flow per
minute compared with laboratory dosage as low as 100 times the
floW per minute. However, it was not believed that Wildwood
inhibitor was concentrated, as a 1:100 dilution against Robena
iron bacteria is still fairly dilute and will probably not be
effective under actual mine conditions.
51
-------
Because of erratic results as to when the inhi-
bitor appears and because it was not effective at high dilu-
tions, it was believed that further testing of Wildwood
inhibitors was necessary. It was hoped that the inhibitor
could be identified, purified, then concentrated so that
only small quantities would be needed to seed the Robena and
Karen mine test sites.
Concentration of Phage-Like Inhibitors by Eluting Lysed
Plates of Iron Bacteria
Three-liter quantities of Wildwood filtrates
specifically inhibitory to Karen and Robena bacteria were
obtained. These filtrates were tested against their speci-
fic bacteria on plates and in liquid iron media and found
to be effective in low dilution as before. These two fil-
trates were designated as the original filtrates and were
kept stored in a deep freeze until aliquots were removed
for further testing and electron microscope studies.
An attempt was made to develop more concentrated
material by plating at low dilution with iron bacteria.
Thirty-six plates were used for each filtrate. After incuba-
tion the plates were eluted using synthetic liquid iron media.
This was followed by low speed centrifugation then millipore
filtration. Again the filtrates (about 200 ml each) were
frozen until used. These transfers (1-1) filtrates were
tested against their specific iron bacteria on overlay
plates and in liquid iron media.
Both the original and 1-1 filtrates were tested
against bacteria on plates. In these tests the filtrates
were progressively diluted from 1:5 up to 1:106 then tested
against the same quantity of bacteria, i.e., 0.4 ml iron
bacteria and 0.1 ml filtrate. It was found that the fil-
trate active against Karen iron bacteria was effective up
to a ratio of 1:100 filtrate to bacterial culture. At this
ratio only a few bacteria grew out on the plates. The fil-
trate active against Robena iron bacteria was less effect-
ive as only a 1:10 ratio inhibited bacteria. The T-l trans-
ferred material showed about the same results. Liquid tube
tests done in triplicate showed incomplete iron oxidation for
six days beyond the controls when using the original fil-
trate against Karen and Robena iron bacteria. The T-l fil-
trates showed incomplete oxidation for only a few days. With
incomplete or partial iron oxidation the liquid portion of
the tube remained mostly clear and the sediment was white
(unoxidized) with a small amount of orange (oxidized) iron.
The iron bacterial controls showed complete iron oxidation
as the liquid became entirely orange and sediment was all
orange.
52
-------
It was not known why the filtrates inhibited iron
bacteria for only six days in liquid cultures. It may be
the ionic environments was not optimum for the adsorption
of phage to their host cells. Adams, p-143 (1959) has stated
that the rate of phage adsorption is depressed markedly by
higher or lower concentrations of salts. The iron bacteria
could also have developed resistance to the phage, or other
inhibitor, which may not be a phage.
Concentration by Ultracentrifugation
Filtrates were subjected to 2 cycles of differential
centrifugation, alternating speeds of 40,000 xg and 2000 xg
for 8 hours. This was done in order to determine if potency
of inhibitors could be increased. This procedure can be used
to purify and concentrate phages. Similar procedures have
been used by Adams, (1959), Bradley, p-234 (1965) and Zimmerer,
(1969). The concentrates were then tested against the iron
bacteria using aliquots of various dilutions on iron agar
overlay plates.
In general, increases in potency were noted with
some exceptions. It was found that a filtrate originally
active against Robena bacteria was increased in potency
from a 1:10 dilution to a i:iO dilution. The filtrate
originally effective against Karen bacteria at a 1:100
dilution was increased to a 1:103 dilution using the con-
centrate. Some filtrates originally show no effect on the
stock culture of F. sulfo , the concentrated material showed
plaques even when diluted to l:l0 . However, no clear areas
of mass bacterial lysis was seen on agar plates. An F. ferro
concentrate gave the same results as the F. sulfo concentrate.
In liquid media however, the concentrated material did not
inhibit any of the strains of bacteria.
Electron Microscope Studies
Aliquots of the Wildwood filtrates were ultra-
centrifuged at 3.7,000 xg for one hour and the residues exa-
mined in the electron microscope. This method has been used
by Zimmerer (1969) to concentrate phages. Photographs were
obtained but no biological agent was detected. Plates con-
taining inhibited iron bacteria were also examined and a
stalked species of bacteria was photographed from the Karen
filtrate inhibitor. This suggested that some genera of
bacteria such as Caulobacter may be inhibitory to iron
bacteria.
Further Testing of Filtrates to Identify Phage-Like Inhibitors
It was strongly believed that every effort must be
made to discover the basis of the inhibitory action of filtrates,
53
-------
particularly from the Wildwood sources. The fact that inhi-
bitory material is heat labile and does not lose activity
by dilution supports the view that it is of biological origin.
The filtrates showing inhibitory action against iron bacteria
as cleared (lysed) areas on iron agar plates were obtained
from neutral mine waters and drainages. The Wildwood and
McCormick mines have been in operation for 20-30 years and
any lime in the rock formation should have been leached out.
Most mine areas become acid after about five years as lime
is leached out. Something has kept the drainages from these
mines neutral and free of iron bacteria, although occasional
samples contain iron bacteria. The iron bacteria-inhibiting
material from these areas may thus be a biological agent.
It was felt necessary to concentrate large quantities of
filtrate from a Wildwood mine area source. Subsequently, an
8 liter quantity of filtrate previously found to be effective
against Robena iron bacteria was prepared from a Wildwood
source. This liquid was concentrated by freeze-drying, then
examined using the electron microscope. It was hoped that
a biological organism could be found. A large quantity of
concentrate was obtained from four-liters of material.
The powder was resuspended in water, centrifuged
at low speed and the precipitate removed. The supernatant
was ultracentrifuged at 37,000 xg for four hours and electron
microscopy done on the sedimented material. Nothing was
seen except amorphous debris.
Aliquots of the freeze-dried powder were inoculated
into various liquid media in order to determine if a micro-
organism could be isolated. No growth was present in media
designed for the isolation of fungi, Streptomyces, iron bac-
teria and sulfur bacteria. The dissolved powder did not in-
hibit iron bacteria In liquid or on agar plates. It was
evident that the freeze-dried powder had no biological
activity and it may be that freeze-drying may have been res-
ponsible.
Testin g for an Autocatalytic Inhibitor
It had been suggested that some of our previous pos-
itive inhibition results with Robena and Karen coal piles
might be due to an autocatalytic inhibitor. This inhibitor
is developed in the death phase of microorganisms; and, when
applied to viable organisms, it caused them to produce more
of the inhibitor and induces death.
Death was induced in all strains of iron bacteria
by homogenizing cultures in a Waring blender with glass beads.
The disrupted cultures were millipore filtered and the fil-
trates tested against fresh cultures of iron bacteria. Testing
54
-------
.wasperformed in liquid synthetic iron solution and on solid
iron agar plates. The results were negative in that the
filtrates did not inhibit fresh iron bacteria in liquid
cultures, nor did plaques or cleared areas appear on agar
plates. The filtrates were tested by themselves in liquid
iron and on agar plates in order to determine if the homo-
genizing and filtering removed and killed the iron bacteria.
None of the filtrates oxidized iron in liquid cultures and
no bacterial iron colonies appeared on agar plates. The
conclusion appeared to be that autocatalytic inhibitors
were not formed in cultures of iron bacteria.
In order to rule out the possibility that filtrates
producing plaques effective against iron bacteria were
caused by an organic product or inorganic effects, a Wild-
wood filtrate previously found to be inhibitory was boiled,
then retested against iron bacteria. No inhibitory plaques
were seen indicating some kind of biological activity was
originally present in the filtrates.
Conclusions
The results tend to disprove the presence of phage
as an iron bacteria inhibitor. Hundreds of samples were
collected from numerous acid and neutral mine areas and
tested for the presence of phage. Iron bacteria inhibitors
were never found in samples obtained from acid mine areas.
Inhibitory effects could sometimes be obtained from drainages
located in neutral or alkaline mine areas, but inhibition
appeared to be caused by chemical agents as filtrates could
not be transferred without losing their inhibiting action.
In a few of the neutral mine areas, drainages prepared as
filtrates appeared to be biological in nature as they could
be transferred by filtration and retained inhibitory acti-
vity against iron bacteria. In most cases, inhibition could
not be proved to be of a biological nature, Although hundreds
of electron micrographs were obtained of filtrates, lysed
areas on agar plates, and various types of concentrated
material, no photographs of phage were ever obtained. The
inhibitory material could be transferred up to a certain
point, was heat labile and could be filtered, By these
methods it could be concentrated but not to a great degree.
As a result of lack of success in great efforts to concen-
trate the material, (ultracentrifugation and elution of many
plates producing only small quantities of inhibitory material),
it is doubtful that large quantities could be produced by
these methods. In addition, the material can only be obtained
at certain times of the year. It is therefore, unlikely
that an unconcentrated natural inhibitor could be economi-
cally applied to mines. However, some electron photographs
of iron bacteria lysed plates were obtained in which Caulo-
55
-------
bacter could be seen. As this microorganism can be filtered,
is harmless and may be the cause of inhibition of iron bacteria,
studies were conducted to find if this microorganism was
inhibitory to iron bacteria on the premise that if Caulo-
bacters act as inhibitors against iron bacteria, they could
be isolated, then easily mass-produced and inoculated into
mines.
56
-------
S E C T I 0 N 12
CAULOBACTERS
BACKGROUND
Previously, electron microscopy showed that Caulobacters
or closely related microorganisms were present on iron agar
plates in which lysis of iron bacteria was seen as plaques. The
plates had been seeded with iron bacteria and filtrates pre
pared from Wildwood and McCormick waters. No Caulobacter or
any other microorganisms except iron bacteria ere seen on the
control plates containing iron bacteria but no Wildwood or McCormick
waters. It was reasoned that Caulobacter or some closely re
lated microorganism might be responsible for inhibitory effects
on iron bacteria.
Caulobacters have stalks which are extermely small in
diameter and can pass through very fine millipore filters. The
diameter of the stalk as given by Poindexter, p- 234 (1964) is
0.2 micron. Zirnmerer, (1969) has reported that Caulobacters
can pass through 0.2 micron millipore filters as he has found
them in electron micrographs. The chances of finding Caulobacters
inotherwise sterile filtrates is particularly good if suction
(negative pressure) is used to prepare filtrates as this tends
to pull the Caulobacters, stalk first, through the membrane f ii-
ter. The microorganisms are harmless and have often been isolated
from lakes, streams, soil, water lines and tap water faucets.
The nature of the holdfast stalk is still unknown, but may
attach to solid objects. They also have a life cycle in which
free swimming forms and stalked forms predominate at one time or
another. If stalked forms can attach to solid objects, there
does not appear to be any reason why they could not attach to
coal; and, since they can live in waters containing only minute
amounts of peptone, there does not seem to be any reason why
they could not live in coal environments. G nera1ly, Caulo
bacters live at neutral pH, but, they could probably be adapted
to a low pH or live in acid waters as part of their life cycle.
This has been reported by Gerencser, (1969). It was also thought
they may attach to iron bacteria as there have been reports in
the literature of this happening to other bacteria, Poindexter,
(1964). Caulobacters may also compete for the food supply and
reduce the number of iron bacteria.
Details of experimental results regarding the static
testing, adaption, tecting under dynamic conditions in the various
microbiological systEms will be discussed in detail in the
following section.
57
-------
Initial Attempts at Testing and Adaptation of Stock Caulobacter
A stock culture of a single strain of Caulobacter,
which normally grows in pond water, was obtained from the Amer-
jean Type Culture Collection and grown in a nutrient solution
recommended by Poindexter, (1964). The liquid standard medium
was designed to allow good growth of Caulobacter but other m Lcro
organisms could also grow. The liquid was defined as standard
medium and is composed of tap water with 0.2% peptone, 0.1%
yeast extract, and 0.02% magnesium sulfate. For solid standard
agar, 1.0% agar was added and allowed to harden in petri dishes.
As originally isolated, this microorganism was obtained from
natural pond waters with only 0.0l%peptone added.
After several transfers in liquid standard medium, this
type III Caulobacter species was transferred directly to the
three modifications of iron media, synthetic I, A and 13.
Stained slides were px epared from liquid aliauots of
the three iron media to which the Caulobacter had be n trans-
ferred. The slides showed what appeared to be detached stalks.
A few non-stalked cells were also present. However, this did not
indicate that Caulobacter was actively growing in the iron media
as these microorganisms could simply have survived as they car-
ried over nutrient from their original growth medium. The Caul-
obacter was tested against all strains of irbn bacteria in the
liquid iron media.
Results showed that Karen iron bacteria were prevented
from oxidizing ferrous iron four days beyond the control tubes
of Karen iron bacteria only. But this delay in iron oxidation
occurred only in iron medium A. A stock culture of F. sulfooxidans
was prevented from oxidizing iron for 9 days in liquid iron
medium A. However, the Caulobacter did not prevent the Robena
iron bacteria from oxidizing iron in any of the iron media in
which it was tested.
Electron micrographs were taken from aliquots of the
tube containing unoxidized iron medium A inoculated with Caul-
obacter and the stock culture of iron bacteria, F. sulfooxidans .
Electron micrographs were also taken of the iron bacterial
control tube containing F. sulfooxidans which had oxidized
iron in medium A.
The electron micrographs were taken from aliquots of
the tube conta 4 όna unoxidized iron medium oρtaining iron
5acteria and Ca1 lobacter. Caulobacter was not see i in aliquots
from the control tube medium containing iron oxidized by the
iron bacteria.
58
-------
The results showed that Caulobacter was not present as
a contaminant in the liquid iron nutrient solution, or as a
mixed contaminant being carried along upon transfer of suppos-
edly pure cultures of iron bacteria. However, the results of
the electron micrographs did not give enough evidence to prove
Caulobacter inhibited iron bacteria in liquid cultures. The
Caulobacters were grown initially in a neutral solution which
was organic in nature, containing peptone and yeast. Upon
direct transfer to the essentially inorganic iron medium, the
Caulobacter cells could have carried over some of the organic
material which may have partially inhibited the iron bacteria.
The stock Caulobacter strain was transferred a sec-
ond time in like iron I, A and B then re-tested in triplicate
against all the iron bacteria, again in the same iron media.
Caulobacter was added to the iron media in a 1:1 and 5:1 ratio
with each specific iron bacterium; i.e., stock cultures of
F. sulfo and F. ferro , Robena (F. sulfo ) and Karen (F. sulfo) .
The dilutions of Caulobacter and iron bacteria used were 1:100
in each synthetic iron medium of acid pH (3.6).
Results showed that Caulobacter prevented Karen
iron bacteria from completely oxidizing ferrous iron for four
days beyond the control tube containing Karen iron bacteria
only. After the fourth day, iron became completely oxidized.
Stock cultures of F. sulfo and F. ferro were prevented from
oxidizing iron for three days. Increasing the ratio of Caulo
bacter over iron bacteria did not substantially prolong delay
of iron oxidation by the iron microorganisms.
Aliquots from the partially oxidized tube of Caulo-
bacter and Karen iron bacteria and fully oxidized Karen bac-
terial control tubes were transferred to iron agar plates.
After growth occurred, Caulobacter could be seen as very small
pinpoint colonies in the heavier growth of yellow iron bac-
terial colonies. The white pinpoint colonies appeared to have
some inhibitory action as clear unoxidized halos (plaques)
appeared around these colonies and also around some of the
iron bacteria. The zones of inhibition on the iron agar plate
containing Karen iron bacteria and unadapted stock Caulobacter
is shown in Figure 17. The control iron agar plate of Karen
iron bacteria without Caulobacter did not show any plaques.
Visual observation of synthetic iron agar plates
containing Caulobacter showed sparse growth of these micro-
organisms which appeared as pinpoint white colonies. Aliquots
obtained from liquid and iron agar plates, and prepared as
stained slides, were examined under the microscope. Caulo-
bacter could not be seen as typically stalked microorganisms.
However, some atypical forms or contaminating microorganisms
were seen. The characteristic appearanφe of Caulobacter
59
-------
FIGURE 17 ZONES OF INHIBITION ON AGAR LAYERS (LEFT) P ND
ELECTRON MICROGRAPH OF CAULOBACTERS (RIGHT)
-------
as seen in an electron microscope (taken from the original
stock culture grown in standard medium)is shown in Figure 17.
In iron medium, detached stalks could be seen. It was not
known at that time if the bacterial form of Caulobacter changed
upon transfer from its original standard medium to iron med
iuiu, if iron in the medium obscured their typical appearance,
or if Caulobacter just did not grow in iron media.
Only one stock culture of Caulobacter had been studied
up to that time. It was hoped that Caulobacters could be
i olated from previously inhibitory waters from Wildwood and
McCormick mines and that they would be inhibitory to iron
bacteria. This will be discussed in the following section.
Isolation of Caulobacter Strains from McCormick and Wildwood
Waters
In an attempt to determine the nature of inhibitory waters,
six strains of Caulobacter were isolated from neutral drainage
sources of Wildwood, and three strains from the McCormick mine
areas. The Caulobacters were grown by adding small quantities
of peptone, (0.01 percent) to the neutral waters, incubating
and plating on standard agar. Colonies grown on agar were
picked and stained to identify; then each Caulobacter strain was
picked and transferred to fresh agar plates to obtain pure
strains. The strains were different as indicated by their
morphology on agar plates and microscope appearance.
All strains of Caulobacter were picked from agar plates
and placed directly into tubes of iron medium A (pH 3.6), incu-
bated and tested against the iron bacteria. The results were
negative in that none of the Caulobacter strains prevent bac-
terial iron oxidation.
Stained slides were prepared of all the isolated Caul-
obacters inoculated into acidic iron media after being trans-
ferred from their naturally neutral waters. The typical stalked
form of Caulobacter could not be seen in the microscope. It
was thought that Caulobacter might have a better chance to adapt
to acid iron conditions if they were to be progressively
adapted to iron media containing more iron and acid since re
suits showed they could not immediately adapt to synthetic
iron media having a much lower pH than their original environ-
ment. This will be discussed in the following section.
Initial Attempt to Adapt Caulobacter Strains to Iron Media
Mixed Caulobacter strains isolated from McCormick, mixed
Caulobacter strains isolated from Wildwood, and the stock Caul-
obacter obtained from the American Type Culture Collection (A.T.C.C.)
61
-------
.
A - Stock ATCC
I
1
B - From McCormick Effluent
FIGURE 18 STOCK AND MCCORMICK ADAPTED CAULOBACTER CULTURES -
STANDARD AGAR PLATES
-------
were added separately to iron medium A. This differed from the
original medium (Table 1 ) in that iron was not added to the
salt medium and the pH was 7.0. The mixed Caulobacter strains
were added to tubes at a dilution of 1:100 in 10 ml of the salt
medium. Peptone was added in the amount of 0.01 percent to
each 10 ml of medium. Tubes were incubated at 270 C for one
week, aliquots placed on slides which were then stained using
a flagella stain to observe stalked microorganisms. All Caul
obacter strains were seen as typical stalked cells.
Transfers were made from the salt medium at pH 7.0 to
a modified iron medium, again at 1:100 dilution in 10 ml of
modified iron medium. The modified medium contained its nor-
mal amount of salts as shown in Table 1 , except that 100 ppm
of Fe II was used instead of the usual 200 ppm. Sulfuric acid
was not used and the pH was 5.0. Peptone was added in the amount
of 0.01 percent. The tubes were incubated for one week, then
flagella stains prepared as before. Microscopic examination
of the stained slides showed mostly bacterial cells appearing
as rods and detached stalks. Only occasional typical stalked
Caulobacter cells were seen.
Aliquots of Caulobacters growing in the modified iron
media were again transferred to the same media, incubated for
a week and stains prepared as before. The appearance of the
Caulobacters was the same as noted previously, i.e., detached
stalks and bacterial cells.
Aliquots from each of the three sets of Caulobacters
(Wildwood, McCormick and stock) were removed and tested against
iron bacteria in the original iron medium, medium A as described
in Table 1 A 1:1 Caulobacter to iron bacteria ratio was used.
Iron bacteria and Caulobacter were diluted 1:100 in 10 ml of the
original iron II medium A (Table 1 ).
Results showed that iron was oxidized in 14 and 8 days
in iron medtLum A (Table 15) inoculated with Karen and Robena
iron bacteria respectively, and McCormick obtained Caulobacter
strains. Iron II was oxi iized in 2 days in all iron bacterial
control tubes containing iron bacteria and media. The negative
controls containing synthetic iron media only showed that iron
II remained unoxidized throughout the test period of 14 days.
Results are shown in Table 15.
Aliquots were removed from all modified iron media
(pH 5.0) containing the Caulobacter strains, and transferred
in the same quantities as before to synthetic iron medium A at
pH 3.6, the composition of which is shown in Table 1 , the
only difference being that 0.01 percent peptone was added. Tubes
were incubated, aliquots removed and stained as before.
63
-------
TABLE 15 - TESTING ADAPTED CAULOBACTERS AGAINST IRON BACTERIA
Oxidation of
Source of Test Iron Fe II (3)
Caulobacters Medium Modifications (1) Bacteria (2) Days
McCormick Strains Synthetic A salts; 100 ppm Karen 14
Fe II, pH 5 Robena 8
Wildwood Strains Synthetic A salts; 100 ppm Karen 7
Fe II, pH 5 Robena 7
A.T.C.C. - Type III Synthetic A salts; 100 ppm Karen 6
Fe II, pH 5 Robe a 3
McCormick Strains Synthetic A, 200 ppm Fe II, Karen 11
pH 3.6 Robena 9
Wildwood Strains Synthetic A, 200 ppm Fe II, Karen 4
pH 3.6 Robena 4
A.T.C.C. Type III Synthetic A, 200 ppm Fe II, Karen 4
pH 3.6 Robena 3
McCormick Strains Synthetic A, pH 3.6 with Karen 9
coal and pyrite Robena 8
Wildwood Strains Synthetic A, pH 3.6 with Karen 2
coal and pyrite Robena 2
A.T.C.C. - Type III Syntheitc A, pH 3.6 with Karen 4
coal and pyrite Robena 3
(1) Details of the iron media compositions have been described in the written commentary.
(2) Caulobacters were grown in their adaption media and tested against iron bacteria in
synthetic A iron media of pH 3.6 as described in Table 1.
(3) The Karen and Robena iron bacterial controls were inoculated with each set of experiments;
Fe II was oxidized in 2 days. The negative controls remained clear, and unoxidized
throughout each test period.
-------
Microscopic examination of the slides showed very few
detached stalks and bacterial cells. The Caulobacters were
tested against Karen and Robena iron bacteria in medium A, pH
3.6 in the same manner as previously described. The results
shown in Table 15 indicate that McCormick strains prevented
Karen iron bacteria from oxidizing Fe II for 11 days and Robena
bacteria for 9 days, beyond the two day Fe II oxidation times
of Karen and Robena bacterial controls. The negative bacterial
controls remained unoxidized throughout the test.
The Caulobacters incubated in the original iron medium
A (Table 1 ) were transferred to the same medium except that
iron was omitted. The medium was acidified to pH 3.5 with sul-
f uric acid. Two gin of coal and 1 gm of pyrite were add d to
each tube containing 10 ml of medium. Coal and pyrite were
added to supply iron and sulfur. After incubation for two
weeks, aliquots were removed and stained as before. Caulobacters
were not seen as cells or stalks. However, coal, pyrite and
iron were present, as debris, to such an extent that very little
else could be seen.
Aliquots were tested against iron bacteria in acid iron
medium A (Table i as previously stated. The McCormick strains
prevented Karen and Robena iron oxidation for about 9 days, Wild
wood strains did not prevent iron oxidation while the A.T.C.C.
strain of Caulobacter prevented Fe II oxidation for only a day
or two beyond the iron bacterial control tubes.
Transfers were made again from acidified medium A with
coal and pyrite added to the same medium at a 1:100 dilution.
After incubation, aliquots were tested against the iron bacteria.
None of the Caulobacter strains prevented bacterial iron oxidation
for more than a few days beyond the iron bacterial controls.
A line sketch of the Caulobacter adaptive procedures is
given in Table 16.
In summary, six combined strains of Caulobacters isolated
from Wildwood waters, and one strain of stock (A.T.C.C.) Caulo-
bacter did not appear to adapt well to acid/iron conditions.
Three combined strains of Caulobacters obtained from the McCor-
mick mine waters showed partial adaption, but small quantities
of peptone appeared to be necessary for adaption to acid/iron
environment.
Inability to adapt may have been caused by transferring
Caulobacters in test tube quantities. It was felt that the strict-
ly aerobic Caulobacters did not get enough oxygen to allow them
to acclimatize to unnatural growth media. In addition, poor in-
hibiting powers may have been caused by testing small populations
65
-------
TABLE 16 - STEPS IN ATTEMPTS TO ADAPT CAULOBACTERS
Iron medium A
H 2 S0 4 added -
Fe II added -
cent peptone
1:100
- pH 3.6
200 ppm
0.01 per
added
dilution
pH 7.3 Mine Waters
containing 0.01 percent
peptone
1:100 dilution
Iron medium salts A
pH 7.0 NoFell-
0.01 percent peptone
added
1:100 dilution
Iron medium A - pH 5.0
100 ppm Fe II added
0.01 percent peptone
added I
1:100 dilution
Iron medium A - pH 3.6
H 2 S0 4 added - 200 ppm Fe
II added -p0.01% peptone
added
1:100 dilution
Iron medium A - pH 3.5
No Fe II added - H 2 S0 4
added - Iron and pyrite
added. No peptone added
1:100 dilution
Iron medium A - pH 3.5
No Fe II added - H 2 S0 4
added. Iron and pyrite
added. No peptone added.
66
-------
of Caulobacters against concentrated cultures of iron bac-
teria. The viable cell counts of iron bacteria tested were
on the order of 1 x i 9 cells per ml. This has been shown
in a previous report by Shearer, R.E. and Everson, W.A.,. p-lO
(1965)
It was hoped that further attempts to adapt Caulobacter
might be more successful if larger flasks and greater quantities
of liquid nutrient medium were used to provide more surface
area.- Actively aerating and constant shaking might provide
better environmental conditions. If full adaption could be at-
tained (with peptone obtained from the acid bacteria or substituted
with the organic content of coal), then further testing would
be conducted in vertical flooded and horizontal systems. These
systems have smaller iron bacterial populations which simulate
natural conditions in a coal mine environment.
Since there were too many individual strains of Caulo-
bacter to be adapted and tested, a decision had to be made as
to how to proceed. The stock culture of Caulobacter was used
as it had shown some initial inhibition against iron bacteria.
In addition, mixed McCormick Caulobacter strains were used
since it was not known which single strain was most effective.
Mixed Mccormick isolated strains were used instead of Wildwood
isolated strains because only three strains of Caulobacter
were obtained from McCormick water, while six strains were ob-
tained from Wildwood water. Also many other types of micro-
organisms were isolated on standard agar plates from Wildwood
waters. It was thought best to avoid other contaminating
bacteria and fungi which might interfere with test results and
give confusing data.
Further Attempts to Adapt Caulobacters to Acid/Iron Environments
The stock culture (A.T.C.C.) of Caulobacter and the
mixed strains of Caulobacters isolated from McCormick neutral
drainages were propagated in neutral sterilized tap water con-
taining 0.01% peptone. These strains were transferred and
gradually adapted to an acid/iron environment in 3-liter flasks
containing only one liter of iron medium to allow a large sur-
face area. Spargers were connected from a sterile air source
and nto the flasks beneath the liquid surface. The flasks were
then shaken and aerated continuously. After several transfers
through gradually increasing concentrations of iron and acid,
the Caulobacters were cultured in a final medium of synthetic
A (Table 1 ) acid mine drainage with the full amount of iron
and a pH of 3.6. It was felt necessary to include small quan-
tities of 0.01% peptone. Gradual adaption of the Caulobacters
to acid/iron conditions were made in a similar manner as prev-
iously described for transferring Caulobacters in test tubes.
67
-------
However, in present studies, larger quantities were used and
cultures shaken and aerated.
These larger quantities of Caulobacters were tested
against Karen and Robena iron bacteria and were found to pre-
vent iron oxidation for about three weeks beyond the iron bac-
terial controls.
The stock (A.T.C.C.) and McCormick isolated Caulobacters
appeared to partially adapt to acid/iron environments; proof of
this will be discussed under Electron Microscope Studies following.
Electron Microscope Studies
The liquid cultures of acid/iron-adapted McCormick and
stock A.P.C.C. Caulobacters used to inoculate the the flooded
vertical coal systems were transferred to standard agar plates.
(The flooded systems will be described in detail later). These
plates are the natural growth medium for Caulobacter. It was
not known if acid/iron adapted Caulobacter baaterial growth
would occur as this medium is organic and has a slightly alkaline
pH. However, excellent growth was obtained from both Caulo-
bacter cultures. These plate cultures were examined in the
electron microscope. Figure 18 shows the microorganisms as
they appear on standard agar plates from the stock A.T.C.C. and
McCormick ironadapted cultures.
The liquid cultures of stock and McCormick Caulobacter
used to seed the coal piles were also examined. These cultures
had been adapted to gradually increasing concentrations of iron
and acid. In addition, a number of successive transfers had
been made in the final synthetic acid drainage. Electron micro-
scope studies showed that the stock and McCormick cultures were
not contaminated. (See Figure 19).
Samples were later obtained from vertical flooded systems
8 and 9. Number 8 has had additions of what was thought to be
ironadapted Caulobacter from the stock AT.C.C. culture, and
from filtered flcCormick mine neutral drainage. Container 9
served as a bacterial control tank and inhibitor was not added.
Samples were withdrawn 50 days after the last addition of acid-
adapted Caulobacter. Since these tanks were dynamic systems
with constant water flow, any washout of inhibitor should have
occurred unless it had become part of the normal flora of the
coal system. The samples were analyzed using the electron
microscope. Various kinds of bacteria were seen and photographs
taken. Sample 9 (control) was characterized by having flagellated
bacteria and crystalline materials. The predominant bacteria
seen in effluents from Sample 8 (test container) were larger than
the flagellated bacteria in the control and appeared in clusters
68
-------
e
f 4
tf -* . q;
if 4 1cr
4
2.
A - Stock ATCC
B - McCormick
FIGURE 19 ADAPTED CAULOBACTER CULTURES USED TO INOCULATE COAL
f
4 IP,,
4 II? %.*
Ip.I
I.
S
S
a
-------
as shown in Figure 20. These bacteria appeared to be the right
size for Caulobacter, 0.51.2 micron, but were without stalks
or flagella normally seen with Caulobacter.
It can be seen that the same bacteria were present on
the agar plates and from the liquid cultures. It also appeared
that the same bacteria were present from effluents of the test
container (#8) after fifty days. In order to determine if the
original stock Caulobacter was truly Caulobacter, as obtained
from A.T.C.C., electron micrographs were made of stock Caulo-
bacter grown on standard agar from its original tap water-peptone
liquid medium. It was found that the original stock Caulobacter
used for adaptation to liquid synthetic iron medium was Caulobacter
as seen in Figure 17.
Further work was aone in attempts to prove that the
stock and McCormick adapted seed cultures were pure (uncontaminated)
and that the same bacteria were present in the cultures used to
seed the flooded container number 8 as in the container itself.
Additional Microbiological Studies on Caulobacter
The stock and McCormick Caulobacter seed cultures
were plated out on standard (Caulobacter) agar, Czapeks(Waksman,
p245 (1967) and Hesseltine, [ 1954]) (Streptomyces and fungi)agar
and iron agar. Good bacterial growth appeared on standard agar,
but scant growth was seen on Czapeks agar, and no growth
recognized as iron bacteria appeared on iron agar.
Aliquots from flooded container 8 (test container) and
9 (bacterial control container) were plated out on the same
media. The same type of colonies was isolated on standard agar
from container 8 as were fdund from the stock and McCormick cul-
tures used to seed the test container. An entirely different
type of bacterial growth was obtained from container 9 on standard
agar.
Aliquots from test container 8 (contains Robena iron
bacteria and Caulobacters) and control container 9 with Robena
iron bacteria only, did not show growth on Czapeks agar.
Iron bacteria from both containers grew as colonies on iron
agar plates. These results appeared to indicate that the same
type of bacterium was present from both seed cultures which
qontained Caulobacter, and, also from container 8, but not 9.
Electron microscope studies of colonies growing on standard
agar plates and isolated separately from the adapted seed cul-
ture of McCormick, and stock Caulobacter as well as from con-
tainer 8 had the same appearance. Short rods, 1 micron ifi
length x 0.5 micron in width were seen. These bacteria were
flagellated, appeared in clusters, but were without stalks.
70
-------
: 4P II
FIGURE 20
TWO ELECTRON MICROGRAPHS OF PREDOMINANT BACTERIA
IN EFFLUENTS FROM CAULOBACTER - INHIBITED SYSTEMS
4,
I
/
i
-------
Summary of Electron Microscope Results
Indications were that the good results in decreased
acidity at the flooded container number 8 were due to the addition
of seed cultures from the stock or McCormick cultures. The
microorganism responsible may be an adapted form of Caulobacter,
although electron photographs do not show the typical appearance
of Caulobacter. Of course, the Caulobacter may have died out
under the extreme acid conditions and another microorganism may
have become predominant.
Proving that the Caulobacters have adapted to such an
extent as shown in the photographs is a long and tedious process
and attempts were not made because of lack of time. It was
believed that vertical flooded-system testing should be repeated
with these cultures in order to clearly prove that they were
responsible for the improvement in iron and acid conditions.
A summary of the major points concerning electron microscopy
done on supposedly adapted Caulobacter cultures and their addition
to flooded vertical coal systems is as follows:
1. Certain of the Caulobacter isolated from the
McCormick mine and adapted Stock Caulobacter
grown both at pH 3.5 and on Standard agar re-
semble some of the bacteria which were found to
reduce iron oxidation and acid formation in coal.
2. Some of the Caulobacter of the original stock
culture also resemble both the McCormick Caulo
bacter and the adapted Stock Caulobacter.
3. The similar appearance of these Caulobacters as
seen with the electron microscope merely helps
to confirm but does not une4uivocally prove that
these are Caulobacter species. Other toxonomic
and characterizing data must be obtained.
Before we can definitely state that Caulobacter act as
inhibitory bacteria, experiments need to be rigorously repeated,
starting with the following:
1. Obtain pure A.T.C.C. cultures of Stock Caulobacter
species.
2. Obtain pure cultures of McCormick Caulobacter from
McCormick water.
These cultures should be obtained pure and kept in their
natural growth medium (tapwater peptone) at slightly alkaline
72
-------
pH. Once pure cultures have been obtained, they should be
again adapted to an acid pH with special care taken to avoid
contamination. Samples should be adapted in triplicate so
that comparisons of the resultant bacteria can be made. Once
adapted to an acid pH, if possible, these cultures should be
tested for their inhibitory properties in carefully controlled
experiments with the vertical flooded coal systems. Experiments
should also be performed in duplicate.
Inoculation of Adapted Caulobacters into Flooded Flowing Vertical
Systems
Inoculations of Caulobacters into media containing iron
bacteria gave some inhibition of growth of the iron bacteria as
evidenced by delay of Fe II oxidation in earlier work. Cells
of Caulobacters were also found previously in inhibited cultures
by electron microscopy. It was of interest to determine if in-
hibition could be obtained in flowing systems having the Fe II
or the suiphide content of coal and pyrite as the nutrient.
Vertical Flooded System
Two containers (8 and 9) similar to those used to study
inhibition of Karen and Robena iron bacteria were set up to
study effects of Caulobacters on the iron bacteria in vertical
flooded systems. The acidproducing bacteria were established
in these flowing systems containing high-sulfur coal refuse and
pyrite as evidenced by iron bacterial viability counts and in-
creased acidity of the effluents. Two hundred milliliters each
of the Caulobacters, sp. Type III from the American Type Cul-
ture Collection and of Caulobacter cultures originally isolated
from McCormick mine water, were added to one of the pairs of
containers (8). These were the ironadapted and batch-produced
Caulobacters which had been grown in one liter amounts in syn-
thetic mine drainage while shaking and aerating. The other
container was free of Caulobacters as ,a control (#9). A second
addition of Caulobacters was made to #8, thirty days after the
first when the pH had risen to 2.9. A third addition was made
205 days after the first and results are shown on Table 17 and
in Figures 21 through 32. Data on sulfates and iron were not
taken beyond 154 days to preserve funds for more extended data
on the significant acidity and pH.
Another test was started later using two containers
(10 and 11). These results are also shown in Table 17 and in
Figures 21 through 32 with some of the data limited to less
than the approximate 250 days on acidity and pH due to paucity
of funds at that time. Evidence of inhibition over at least
some period of time is seen both in chemistry and microbiology
of the systems. Statistical analyses of the data snowi.ng in
73
-------
TABLE 17 - VIABILITY OF ROBENA BACTERIA
IN VERTICAL STREAMS WITH AND WITHOUT CAULOBACTER INHIBITOR
Cells/mi
Days After Adding #8 #9
Caulobacter Caulobacter No Caulobacter
0 30,000 1,300
2 7,900 10,000
9 10,700 8,000
17 30,000 30,000
23 2,000 3,000
30 100 2,000
37 100 5 000
45 2.900 3,500
51 3 600 55,000
58 8:000 43,000
65 700 10,000
72 35,000 150,000
80 2,200 23,000
88 800 8,000
94 3,000 5,000
102 1,400 10,000
109 1 ,000 2,000
116 6,000 8,000
123 1,700 9,200
129 1,800 3 000
137 300 1,000
144 700 -
151 300 <1,000
158 500 5,000
164 200 15,000
172 300 2,000
178 400 2,000
186 200 900
193 <100 1,300
200 <100 1,000
74
-------
TABLE 17 VIABILITY OF ROBENA BACTERIA
IN VERTICAL STREAMS WITH AND WITHOUT CAULOBACTER INHIBITOR
(conti nued)
Days After Adding
Caulobacter
6
2
9
16
23
29
37
44
51
58
64
72
78
86
93
100
#11
Caul obacter
1,180,000
670,000
4,000
120,000
300,000
300,000
328,000
10,000
17,000
<1 ,000
2,500
< 100
< 100
1 ,700
1 ,900
< 100
#10
NO Caulobacter
1,950,000
1,000,000
5 ,000
80,000
58,000
30,000
43 ,000
10,000
8 ,000
64,000
5,000
6,000
3,000
25,000
2 ,500
2 ,000
3
U i
Cells/mi
-------
I I I I I II I I II I I
3 .
p l11C u1o5actar
$1.0 \
Contr 1
I I I Figure 22 - Test 1
Days After Adding Caulobacter
Figure 21 - Test 2
Effect of Caulobacter on Production of
Ferrous Iron by Robena Bacteria
65
60
5s
45
401
25
23
5 J
0
1.10 120 130 140 150 160 170
Days After Adding Caulobacter
-------
igure 23 - Test 1
10
Coz tro1
I i I
Days After Adding Caulobacter
Figure 24 - Test 2
Effect of Caulobacter on [ roduction of
Total Iron by Robena Bacteria
- .4
- . 4
lS S.P9 Control
50
0
0
4J
0
10 20
811 Cau.Lobact.r
Days After Adding Caulobacter
40 50 60 70 80 90 100 110 120 130 140 150 160
0 10 20 30 40 50 60 70 80 90 100
-------
c v
4 )
U
4-4
C )
Effect of Caulobacter on Sulfate Production
I. y Robena Bacteria
Figure 25 - Test 1
Days After Adding Inhibitor
Figure 26 - Test 2
co
3000
c v
4 .) 2000
(U
1500
Cl) 1000
0 10 20 30 40 50 60 70 90 90 100 110 120 130 140 150 160
Days After Adding Caulobacter
10 20 30 40 50 60 70 90 50
-------
I I I I
/
- .4
Ό0
Control
II1tI1IIlIIIIIII!IIIIIlItIII IIItIIIIIIIl I IIIIi1
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
Days After Adding Caulobacter
Figure 27 - Effect of Caulobacter on Oxidation Times of Robena Bacteria
Test 1
-------
I I I I I I 1 1 =
- - - - -
Q 411 Caulobacter
I
\ _o
tr \ ,O -
110 Control
0IIII ILIIII I1IIIIIIIiIIIItIIIII1IIII4
0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 120 136 1 4
Days After First Addition of Inhibitor
Effect of Caulobacter on Oxidation Times of Robena Bacteria
Days After Adding Inhibitor
Figure 29 Test 2
Effect of Caulobacter on Acid Production Ly oL a )actc ria
10
0
-I
U
2
.4
C
2
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
U J
-------
7000
6500 ,
000
i1!hIhIhIhIh(tIh11l I
Days After Adding Caulobacter
Figure 30 - Effect of Caulobacter on Acid Production by Robena Bacteria
Test 1
-η
5500
5000
4500
co
L 400 °
3500
a
3000
0 2500
. :; 2000
0
iooo
S S
119 Control
500
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
-------
.1 I I I I 1 1 1 I I I I I QI
3.1 ii
0
I,;
3.2 /
31 . .
9-. ,. 0
16 C.strei
d s-- .4
3.6
2.11:
2.2 I
1.I
I I I I I I I I i I I I i I I I i I I i I i Ii I I I i I ii II
1.1 I
* 20 36 40 00 110 20 60 60 100 110 130 130 140 130 160 170 100 1 0 200 21S 220 230 340 230
Days After Adding Caulobacter
Figure 31 - Effect of Caulobacter in pH of Cultures of
Robena Bacteria
Test 1
82
-------
6.
Days After Adding Inhibitor
Effect of Caulobacter in pH of Cultures of
Robena Bacteria
6.
a
3.6
3.4
3.2
3.0
2.0
Oil Caulobacter
2.6
2.4
2.2
2.0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Figure 32 - Test 2
83
-------
hibition are given in Appendix 1.
A substitute for peptone must have been present in the
flooded vertical coal systems as any initial peptone added with
the Caulobacters would have been washed out in these flowing
systems.
It seemed advisable to compare the number of viable
Caulobacter cells from natural sources (Wildwood and McCormick
waters) with those of the more concentrated McCormick and stock
acid/ iron adapted strains.
Caulobacter Viability Studies
This work involved studies to determine the concen-
trations of viable cells of the adapted McCormick and stock
Caulobacter strains. Comparisons were also made to determine
the number of naturally occurring Caulobacter from McCormick
and Wildwood mine area waters. Standard agar was used as a
plating medium. Since a differential medium for Caulobacter
does not exist;i.e., a medium which allows only Caulobacter
to grow, accurate counts canndt be obtained from the mine
area waters. However, estimates of the total number of viable
cells can be made of a variety of microorganisms which can be
grown on this plating medium. Many soil and water microorganisms
are present in neutral mine waters which can grow on the
standard medium. Fairly accurate Caulobacter counts can be
obtained from the stock and adapted Caulobacter as these are
pure cultures. The stock and McCormick strains of Cΰulobacters
were grown in a synthetic mine drainage. Therefore, the same
sterile mine drainages were used to prepare serial dilutions
which were subsequently plated on standard agar. The Wildwood
and McCormick waters were serially diluted in tap water with
0.01% peptone. This is commonly used to isolate Caulobacter
from natural sources. The inhibitory waters were about one month
old and filtrates from these waters had shown inhibitory plaques
against Robena iron bacteria on iron agar plates. If natural
sources are allowed to stand for any length of time, naturally
occurring Caulobacters tend to increase in numbers. Poindexter,
p-234 (1964) has stated that Caulobacter numbers increase with
age in water supplies. These drainages, therefor. should con-
tain Caulobacter and have shown some inhibitory power against
iron bacteria.
Serial dilutions were made, aliquots plated and incu-
bated. Counts were made on plates showing viable cells after
three days of incubation. The stock and McCormick adapted
Caulobacter were on the order of 1012 viable Caulobacter cells
per ml. Two waters from Wildwood and two from McCormicK all
gave viability counts on the order of l0 cells per ml. It
84
-------
should be noted that this number represents many types
of microorganisms capable of growing on this medium. The
number of viable Caulobacter colonies only would be lower.
The results show that the stock adapted Caulobacter
strains are roughly 106 times greater in number than the natur-
ally occurring strains. The 1012 cells of adapted Caulobacter
is by no means an optimum number as greater cell yields can no
doubt be obtained by using sedimented or supernatant cells.
The results suggested that once optimum cell yields can be
obtained, then it becomes feasible to inoculate mines with
massproduced packed cell cultures.
Initial Work with New Strains of Caulobacter
In accordance with the suggestions of the contract
monitor, contract was made with Dr. Vincent Gerencser, con-
sultant on Caulobacters. Dr. Gerencser suggested we test
strains of Caulobacters which have lived in acid environments.
These strains may easily adapt to acid/iron environments and
possibly show some inhibition against the iron bacteria in
their natural coal environment.
Four strains of Caulobacter vibroides were obtained
from Dr. Gerencser. These strains were labeled C.V. 143, 144,
145 and 146. Although the source of these strains was a lake
which has had a pH as low as 4.0, the strains were isolated at
a later date when the pH of the lake water was 6.5. These
strains appear to have a life cycle which permits them to live
at different pH values. Since they have been isolated from
waters having lower pH values, they appear to be good candidates
for adaption to acid mine drainage.
Adaption of Caulobacters in Mine Drainage
The four species were stained and observed in the micro-
scope for later comparison with the same strains adapted to an
acid iron environment. The four strains appeared as typical
stalked forms. The strains were transferred from the initial
proprietary growth medium designed by Gerencser (1969) to the
same medium adjusted to DH 7.0 and 4.0. This growth medium
does not contain iron. Transfers were also made to a synthetic
acid mine drainage without ferrous iron but adjusted to pH 3.9
using sulfuric acid. Additional transfers were made to synthetic
acid mine drainage containing ferrous iron and adjusted to pH
3.6 with sulfuric acid. All transfers were made in duplicate
in flasks which were then incubated while being shaken contin-
uously to obtain optimum growth.
After six days, all flasks showed visible growth in
85
-------
Gerencsers medium at both pH 7 and 4. One strain showed visible
growth in synthetic acid mine drainage with and without ferrous
iron. All four strains showed visible growth in liquid acid syn-
thetic mine drainage, as evidenced by clouding of the medium.
However, ferrous iron was not oxidized, indicating their growth
was not at the expense of oxidizing iron. The nutrient source
was iron medium A as described in Table 1 . All strains were
transferred 10 times in liquid acid synthetic mine drainage and
continued to show good growth.
Aliquot from the Caulobacter vibroides (C.V.) series
143 through 146 grown in Gerencsers medium and also in syn-
thetic acid mine drainage, were plated on standard agar. The
C.V. colonies plated from Gerencers medium were white, confluent
and appeared to have a slime coating. The C.V. colonies plated
from synthetic acid mine drainage were greywhite, raised,
glistening and appeared smaller than the colonies obtained from
Gerendsers medium. The acid/iron adapted C.V. series colonies
appeared to be similar in appearance to the colonies obtained
from acid/iron adapted stock and McCormick Caulobacters.
Static Testing Against Iron Bacteria
The four C.V. strains, after having been grown in
synthetic iron medium A., were tested against Robena iron bacteria,
also in synthetic A and I acid iron media as described on Table
1 . A 1:1 ratio of Caulobacters to Robena iron bacteria were
diluted 1:100 in the iron media. Three of the four C.V. strains
caused a delay of two weeks in the oxidation of the Fe II by
Robena iron bacteria in mine drainages I and A. The iron bac-
teria control tubes oxidized Fe II in two days, while the Fe II
in the negative control tubes containing synthetic mine drainage
only, remained unoxidized.
Testing the C.V. strains against Robena iron bacteria
on iron agar plates showed that iron colonies were partially in-
hibited as ihdicated by clear areas on the plates.
The results showed that the tour new strains of Caulo
bacters [ C.V. series] were readily adaptable for growth under
acid mine conditions and partially prevented iron bacteria from
growing.
Some initial work was done in setting up vertical flooded
systems for testing the newly obtained C.V. series strains of
Caulobacters. However, work was stopped because of depletion
of funds.
86
-------
Flooded Horizontal System
To determine if Caulobacters could inhibit throughout
a horizontal system when introduced only at the influent end,
one liter of Caulobacter culture was introduced into one of the
troughs used previously to test natural inhibitor. Results
are shown in Ta1 le 18. The evidence, in general, is that there
is effective inhibition and that the inhibition was moving
downs treani.
Ferrous iron levels at sample ports 3-2, 34 and 35
fell from the 30s to the range 0 to 10 ppm somewhat gradually,
more rapidly in 3-2 and less so in 3-5, suggesting downstream
travel of inhibitors. Somewhat similar conditions developed
with sulfates, viabiljtjes, acidities and total iron. pH failed
to improve in port 3-5.
A second trough was readied for a replicate test, but
this was not used because of depletion of funds.
87
-------
TABLE 18 - EFFECTS OF CAULOBAcTERS ALONG HORIZONTAL SYSTEMS
02 03
No With
imh ibitor Caulobacter
2 1 22 24 25 29 3 1 32 34 35 3 9
Day. After
Adding
tnhibitor
to #3
Ferrous
Iron
(ppm) 1 0 0.1 15 14 41 0 35 39 38 21
6 0 2.5 33 2.8 101 0 15 111 40 109
13 0 8 50 29 52 0.1 36 132 60 135
20 0 1.8 40 7 70 0 16 130 0 200
27 0 1.1 15 3.8 47 0 0 4.5 0 54
34 0 1 30 7 31 0 0 13 11 63
41 0 5 29 23 53 0 35 220 265 264
40 0 0 0.2 0.2 6 0 0 60 21 41
56 0 3 3 16 28 0 0 6 28 42
62 0 8 29 6 65 0 0 0 26 60
68 0 6 46 22 32 0 0 0 27 25
76 0 15 25 10 16 0 0 0 10
Sulfates
(ppm) 1 1000 125 160 375 850 150 710 1100 875 1025
6 125 250 425 475 1025 125 600 1000 625 1150
13 150 175 400 600 1075 250 700 1000 600 1250
20 125 100 425 550 1000 125 200 750 250 1475
27 145 350 150 400 850 100 100 600 145 1000
34 125 275 425 500 850 150 150 700 425 1650
41 150 300 150 100 1125 140 275 1000 1025 1700
48 60 30 90 175 600 90 100 725 275 1325
56 175 173 50 40 625 100 125 250 250 1075
62 125 425 265 300 850 125 50 175 125 1325
68 125 275 125 125 710 100 140 100 125 1125
76 120 100 250 300 410 100 100 150 225 1116
Viability
(thousand.
of
cells/mi) 0 2.5 30 0.8 30 8 10.9 30 30 30 9.5
8 5 30 30 30 30 3.5 14.5 28 34 4.6
15 3.3 30 21.5 30 30 2.0 18.5 22 78 240
23 3 8 12 14 6 1.3 0.4 1.4 10.8 1.2
29 2 4 17 20 4 1 0.1 1.6 2.3 5
37 2 1 7 300 10 2.5 0.1 13 3.4 4
43 3 35 16 80 15 4,5 0,7 20 35 20
50 7 28 8 40 13 .5 0.1 0.5 0.3 2.8
57 18 13 10 74 36 6.3 0.1 0.4 3.9 5.8
64 2 12 11 26 10 10 0.4 0.5 4.3 2.5
71 0.9 1.3 1.3 30 5 1 1 1 4 1
79 2 70 32 300 105 1.8 1.8 2.1 2.8 30
-------
TABLE 18 - EFFECTS OF CAULOBACTERS ALONG HORIZONTAL SYSTEMS (contd)
pH 1 4.45 3.72 3.35 3.2 2.87 4.0 3.6 3.65 3.85 4.82
6 3.5 2.65 2.5 2.42 2.15 3.62 3,3 3.05 3.1 2.85
13 3.7 2.9 2.6 2.5 2.3 3.75 3.7 3.05 3.15 2.85
20 3.72 3.1 2.8 2.65 2.4 5.9 3.9 3.05 3.15 3.0
27 4.35 2.75 2.9 2.6 2.3 5.95 6.2 3.7 3.4 3.2
34 3.7 2.7 2.6 2.55 2.3 3.95 6.3 5.1 3.1 2.95
41 3.5 2.7 2.7 2.5 2.2 3.8 4.88 2.85 2.55 2.95
48 3.7 2.7 2.9 2.6 2.4 4.2 3.78 3.25 2.9 3.15
56 3.95 2.8 3.15 2.75 2.35 4.42 5.8 6.0 3.1 3.0
62 4.95 2.6 2.75 2.6 2.25 3.95 5.3 6.2 3.0 3.1
68 5.55 2.7 2.6 2.6 2.4 4.4 5.55 6.4 3.05 3.05
76 5.45 2.95 2.8 2.6 2.45 5.75 6.05 6.8 3.35 3.10
Acidity
(ppm) 1 30 109 320 545 1385 55 355 375 395 190
6 40 310 430 625 1695 55 205 515 375 820
13 20 155 390 605 1530 20 130 485 310 745
20 25 105 345 550 1615 5 75 445 150 770
27 20 250 160 490 1050 20 5 70 60 360
34 25 335 380 420 1040 20 15 55 175 575
41 45 255 255 485 1230 25 80 595 835 770
48 35 215 160 365 720 15 14 30 255 365
56 30 230 80 300 755 16 2.5 20 165 540
62 14 410 235 300 1010 26 15 61 172 700
68 4 295 305 435 855 10 6 10 153 662
76 12 150 230 420 700 3 1 16 116 550
Total
I ron
(ppm) 1 5.2 12.2 47 97 271 7.8 90 106 109 53
6 0 39 60 144 337 0 24 129 53 154
13 1.8 16 61 104 305 1.6 38 154 68 202
20 0.1 10 53 91 353 0 21 161 8 238
27 0.2 36 24 90 228 0.1 0,2 12 31 70
34 2 56 67 68 240 0.1 0.1 28 43 168
41 2 42 48 97 323 3 41 256 314 351
48 26 31 26 64 153 26 7 106 74 136
56 1 40 9 57 164 3 5 14 36 184
62 2 84 40 53 257 5 2 3 38 276
68 0.5 55 63 70 190 1 1 6 35 281
76 0 21 37 83 73 0 1 5 15 223
k refer to sample ports in numerical succession starting
-------
SECTION 13
STREPTOMYCES AS PRODUCERS OF ANTIBIOTIC INHIBITORS
Theoretical Approach
Microorganisms of the order Antinomycetales (especially
Streptomyces which produce antibiotics) are found in the soil and
could conceivably be present in neutral drainage. The waters from
Wildwood and McCormick areas might contain antibiotic-producing
bacteria and resultant antibiotics could kill iron bacteria. Sam-
ples of these waters were plated on Czapeks agar media but so
many molds were present that any growth of the Streptomyces might
have been obscured. A thorough literature search was not made to
determine if the antibiotic-producing Streptomyces could produce
antibiotics under stressed conditions as found in acid mine drain-
age, but a brief review of the bacteriological literature did not
reveal any attempts to perform work of this nature.
It was proposed that antibiotic sensitivity disks first be
obtained and tested against all strains of iron and sulfur bac-
teria. If effective against the iron bacteria, the microorg nisnis
producing these antibiotics antagonistic to iron bacteria could be
obtained and adapted to the environmental acid/iron conditions of
iron bacteria. It was hoped that mutant or adapted strains of
certain Streptomyces could be induced to grow in acid mine environ-
ments and continue to produce antibiotics which would kill off the
iron and sulfur bacteria.
Antimicrobial Sensitivity Disk Testing
Various selective antibiotic disks were obtained and tested
against the five strains of iron bacteria as shown in Table 19. The
selective disks contained antibiotics produced by Streptomyces
species which might conceivably be present in the soil of mine en-
vironments. However, a detailed study was not made to determine the
pH of the various Streptomycesproducing antibiotics. Initial test-
ing was done only to determine if any of the antibiotics inhibited
the iron bacteria. Disks can be obtained in various dosage levels
but, for our p .irposes, it was believed that disks should be tested
at the lowest available concentration since future testing in mine
areas would be limited to crude extracts of antibiotics to avoid
high cost, or the use of antibiotic-producing strains of bacteria
or possibly adaption of the same bacteria to acid mine conditions.
In either case, the bacteria or antibiotic would not be as potent
as highly purified antibiotics obtained from drug companies.
Plate testing was done by inoculating the solid iron agar
in soft agar overlays. Each iron bacterium was tested separately.
91
-------
Table 1.9 - AntibiotiC Sensitivity Disk Testing
Zflectivenesi on
_____ Solid tron M.dia(2) Liquid Iron Media(3 )
Antiobiotic MicroCl) Produced Commercial F. F. T. P F. T.
Disk gram By Availibility Sulfo Ferro Ferro Karen Robena T.thio Sulfo Ferro Ferro Karen Robena T.thjo
Novobiocth S S. Niveus 40 40 40 50 Clear 50 I I I I I I
S. Spheroid.. Yes
Methacyclin. S S. Rimosue ft 2 2 ft ft ft R ft ft R R R
Disi. thy Ichor-
tetracycline ft ft 2 R ft ft K R K K R K
Neomycin s s. rradie ft R ft ft R ft R R ft K K R
Kanamycin 5 S. Kanamyceticus R 2 R ft R R K R R K K R
(Kantrex)
Lincbmycin 2 S. Lincolnesis R ft ft ft ft R ft R R R R R
Oleandomycin 2 S. antibio- Yes 1 1 1 NC 3 K ft R R K K R
ticus
I. Tetracycline S S. species 2 1 2 NC 5 R ft K R R K K
Chlorotetracyline 10 S. Aur.o Ye. 2 2 2 2 2 30 R R K K K I
(Aureomycin) faci.ns
Bacitracin A. 20 B. Subtilis 1 R 2 2 2 R K R R R R R
Dihydro-
streptomycin 2 ft R R R ft ft K R K R K R
Oxytetracycline 10 S. ftimosus Patented 2 2 2 NG 4 ft ft H R R R R
(Terx amycin)
Str.ptomycin 2 ft ft I. NC 1 R K ft K R R R
Nystatin 1000 S. noursei Restricted 3 4 5 NC 7 K R ft R R R R
Penicillin 20 R R ft 5 5 R R K K R R R
(1) These were the lowest available dosages which could be obtained.
(2) The zones of bacterial inhibition were measured from the edge of the disk and across
the annulus of the clear area in millimeters. K - Bacteria were resistant. I - Iron did
not become oxidized. NC No bacterial growth.
-------
After the agar hardened, each disk was placed onto the agar
using sterile techniques. The plates were incubated until bac-
terial growth was evident and the zones of inhibition measured
across the annulus of the clear zones.
Although not designed for use in liquid media, each type
of disk was placed into 10 ml of synthetic iron media after first
inoculating with iron bacteria. Each disk was tested individually
against each strain of iron bacteria inoculated into synthetic
I iron medium. Positive bacterial controls of ea:ch strain of
iron bacteria, as well as negative controls, were included with
each series of tubes tested. The tubes were incubated and observed
daily for the appearance of oxidized ferrous iron.
Results are shown in Table 19. It was very clear that the
antibiotic novobiocin was extremely effective at low dosages against
all the strains of iron bacteria. On solid media, the zones of
inhibition were very large and clear. No secondary satellite
bacteria were seen. This indicated bactericidal action rather than
just a bacteriostatic effect. Novobiocin also inhIbited the bac-
teria in liquid media as evidenced by the tubes remaining clear for
various lengths of time. The tube containing F. sulfo was clear
for nine days after which it became partially oxidized and remained
so for 41 days. F. ferro , Karen and Robena tubes remained per-
fectly clear for 41 days. The only strain against which novobiocin
was not extremely effective in liquid media was 2 . ferro . The tube
remained clear for ten days, after which it became oxidized as
compared with the mere two-day inhibition of the control tubes.
After 41 days, the negative control tubes containing syn-
thetic iron medium,but no bacteria,became partially oxidized. This
indicated the beginning of chemical iron oxidation.
The excellent results obtained with novobiocin suggested
that further studies be done with the antibiotic-producing micro-
organisms. Streptomyces niveus and Streptomyces spheroides . Novo
biocin is an acidic antibiotic which consists of three moities:
a sugar, a coumarin and a substituted phenol. Its action may be due
to one of these compounds when used at pH 3.5 with the iron bac-
teria. However, rather than typing a chemical approach using var-
ious coumarins and phenol&cs, the bacteria producing this anti-
biotic were obtained and tested.
In summary, the antibiotics tested included novobiocin,
methacycline, dimethychiortetracycline, neomycin, kanamycin,
Kantrex, lincomycin, oleandomycin, tetracycline, chlorotetra
cycline, Aureomycin, bacitracin, dihydrostreptomycin, oxytetra-
cycline, Terramycin, streptomycin, nystatin and penicillin. All
antibiotics were tested as disks, except for bacitracin and pen-
icillin, and are produced by various species of Streptomyces. The
93
-------
most effective antibiotic was novobiocin which killed all
strains of iron bacteria, both in acid liquid synthetic iron
medium and on iron agar plates. Two other antibiotics,
oleandomycin and Aureomycin, showed inhibitory effects on
plates but not in liquid.
An attempt was made to isolate the sulfuroxidizing
bacterium T. thiooxidans from flooded vertical coal pile
systems. This was done since this bacterium is also involved
in the oxidation of pyritic materials and an inhibitor might
be found to be effective against it. Aliquots from all
flooded vertical systems with Robena and Karen were placed
into sulfur and thiosulfate broths at low dilution, 1:10.
After three weeks of incubation, these cultures were diluted
1:100 with fresh nutrient broth. Higher dilutions had to be
made to guard against false positive results due to chemical
oxidation of sulfur. Oxidation of sulfur and thiosulfate
solutions is a characteristic of the sulfur bacteria and is
determined by clouding of the media with adrop in pH. The
T. thio was isolated from flooded vertical system number one.
Number one is the Karen bacterial control system containing
bacteria fron the Karen mine area. At the time the original
aliquot was taken, the p11 of this pile was 4.5 It was in-
teresting to note that T. thio was not isolated from the other
systems. It may be that either the pH was too high for the
bacterium to be viable or this bacterium was never present
after isolation on thiosulfate agar plates, antibiotic sensi-
tivity tests were performed to determine if this bacterium
was sensitive to any of the antibiotics.
Antibiotic disks were tested against the T. thiooxidans
on solid thiosulfate agar plates and in a liquid thiosulfate
medium. This organism grows as nearly transparent colonies
on thiosulfate agar. Large number of colonies cloud the agar.
In liquid thiosulfate, growth is characterized by a uniform
turbidity; also, sulfur is precipitated with the medium
becoming more acid.
The disks were aseptically added to heavy inocula
of T. thio on thiosulfate agar and liquid medium. The results
are shown on Table 19. Tubes and plates, along with control
tubes, were then incubated until growth occurred in the con-
trol tubes and plates. It can readily be seen that only two
antibiotics were effective against T. thio ; these were novo-
biocin and Aureomycin. The sulfur bacterium was sensitive
both on plates and in liquid media. These same two antibiotics
were previously shown to be effective against the iron bacteria.
Therefore, if the Streptomyces producing these antibiotics
are active against the iron bacteria, they will also inhibit
the sulfur bacteria.
94
-------
Initial Testing of Antibiotic-producing Bacteria
Since some antibiotics were inhibitory to iron bacteria,
the bacteria producing these antibiotics were obtained. Tests
were conducted in order to determine if these special antibiotic
producing strains of Streptomyces could inhibit iron bacteria.
The antibiotic producers, S. niveus and S. spheroides (novo-
biocin), S. antibioticus (olean omycin), and S. aureofaciens (Aureo
mycin), were each grown in 100 ml quantities of media commonly used
to grow these bacteria, i.e., Czapeks, (Hesseltine, p142 (1959))
obtained from Balitmore Biological Laboratories, and cporulation
broth, American Type Culture Collection Catalogue, p-l30 (1968).
Sporulation broth is a complex organic liquid composed of yeast ex-
tract, beef extract, tryptose, glucose and traces of ferrous sulfate.
The pH was 7.2. The Streptomyces species did not show early growth
in Czapeks medium which is less complex than sporulation broth, and
is composed of sucrose, sodium nitrate, dipotassium phosphate, mag-
nesium sulfate, potassium chloride and ferrous sulfate. This medium
has a pH of 7.3. Both fermentation liquids are about neutral in
pH and contain sugars used for metabolism of heterotrophic bacteria.
The Streptomyces were grown in flasks by shaking, using aeration to
obtain better growth. After one week, good growth of all the
Streptomyces species was obtained in sporulation medium but not in
Czapeks nutrient solution, but after forty days good growth of
the Streptomyces species was obtained in Czapeks solution.
After good growth was obtained, crude filtrates were prepared
in order to remove dbbris. Filtrates were prepared by filtering
the fermentation broths through Whatman filter paper. The fil-
trates were then tested against iron bacteria within a few
hours after the filtrates were prepared. The filtrates were dil-
uted 1:100, 1:1000 and 1:10,000 in synthetic iron solution I as
given in Table 1 , then the strains of iron bacteria were added.
The results are shown in Table 20. The final dilution of
filtrate in the synthetic iron was 1:100. Higher dilutions did
not inhibit any of the iron bacteria. It was interesting to note
that incomplete iron oxidation occurred in many tests and stayed
at that point, never becoming completely oxidized. This is an in-
frequent occurrence, as in most cases incomplete bacterial oxi-
dation is followed in one or two days by complete ferrous iron ox-
idations. It should be understood that the crude filtrates were
diluted 1:100 in syntehtic iron media and were used against cultures
of iron bacteria normally producing on the order of 107 to 108
viable cells per ml. In addition, the growth conditions for the
Streptomyces were not optimal; i.e., special growth factors were
not added as information about them is proprietary to the drug
manufacturers. The results tend to show that even crude- cultures
of the effective antibiotic producing Streptomyces strains in-
95
-------
TABLE 20 - TESTING ANTIBIOTIC PRODUCER PRODUCTS
AGAINST IRON BACTERIA
Oxidation of Oxidation of ferrous iron
Iron ferrous iron bacterial control tubes Inhibitory
Streptomyces species bacteria in days days 4 effect
S. aureofaciena F. aulfo 2 2 None
- P. ferro 2 2 None
T. ferro 2 2 None
Karen 4partial 2 5complete 3 2 Partial
Robena 2 2 None
S. antibioticus F. aulfo 10partial 2 not contpl te
in 21 2 Good
F. ferro 10 2 Good
N T. ferro 7 2 Fair
Karen 14 2 Fair
Robena 14partial 2 17-complete 3 2 Good
s. niveus F. sulfo 10partial 2 not complete
in 21 2 Completely
inhibitory
F. ferro 5partial 2 21-complete 3 2 Excellent
T. ferro 6 2 Fair
Karen not oxidized in 21 days 2 Completely
inhibitory
U Robena 5partial 2 21-complete 3 2 Excellent
S. apheroides P. sulfo 5partial 2 17-complete 3 2 Excellent
F. ferro 5partial 2 17-complete 3 2 Excellent
It N T. ferro 4partial 2 7-complete 3 2 Fair
II N Karen not oxidized in 21 days 2 Completely
inhibitory
N N Robena 4partial 2 6complete 3 2 Fair
2 In partial oxidation, the liquid portion of tube is clear and sediment is white
(unoxidized iron) with a small amount of orange (oxidized) iron.
3 In complete oxidation, the liquid becomes entirely orange and sediment is all
orange.
Oxidation results were obtained using aliquots grown in sporulation broth. Later
aliquots from Czapeks broth showed oxidation times to be on the same order of
magnitude.
-------
hibited iron bacteria. However, it was not known if the anti-
biotic-producing Streptomyces bacteria produced antibiotics in
their natural growth medium which subsequently inhibited the iron
bacteria upon being transferred to an acid iron medium, or if the
Streptomyces were growing and releasing antibiotic in the acid
iron medium.
In order to determine if the Streptomyces were growing
in the acid iron media, aliquots were removed from the inhibited
iron bacteria tubes and plated on iron and Czapeks agars. No
Streptomyces species were seen on the iron and Czapeks agars.
Thus,it appeared the Streptomyces did not grow on iron medium.
Tests were made to determine if it was the Streptomyces
bacteria, or their antibiotics products,which were responsible
for inhibition. Aliquots were removed from the sporulation
medium of the actively growing StreptQxnyces species. The aliquots
were divided in half, with one portion added directly to acid syn-
thetic mine drainage. The other portion was filtered, using
Whatman paper, then millipore filtered for sterilization. The
sterile filtrates were added to a second series of tubes con-
taining acid synthetic mine drainage. Each of the strains of iron
bacteria was added separately to the tubes. After incubation,
it was found that both the filtered and unfiltered aliquots in-
hibited the iron bacteria. It appeared that it was the anti-
biotics rather than the bacteria responsible for inhibition. Ad-
ditional tests were made in which Streptomyces aliquots were
millipore filtered, the filter pad washed with sterile water,
and the filter itself added to tubes of acidic synthetic mine
drainage. The iron bacteria were added and the tubes incubated.
It was Sound that the Streptomyces retained on the filters also
inhibited the iron bacteria. However, the tubes did not cloud
nor show any signs of bacterial growth indicating that the
Streptomyces did not grow in the synthetic mine drainage. The
conclusion appeared to be that the growth and antibiotic pro-
ducts produced by the Streptomyces are closely bound and it is
difficult to separate the Streptomyces bacteria from their anti-
biotic products. In spite of the fact that the millipore-retained
Streptomyces bacteria inhibited iron bacteria, no growth of the
Streptomyces was observed in tubes of synthetic mine drainage;
therefore, the Streptomyces did not adapt to the iron media under
those conditions. It would be correct then to state that anti-
biotics produced by certain Streptomyces species inhibited iron
bacteria in synthetic acid mine drainage; however, the species
will not grow in acid mine drainage.
Partial Adaption of Antibiotic-producing Streptomyces
Since adaption of the Streptomyces species did not
occur upon direct transfer to synthetic acid mine drainage,
97
-------
attempts were made to gradually adapt these species to coal mine
environments. Initial attempts involved gradually decreasing
the sugar content and increasing the iron and acid concentration.
Sporulation arid synthetic iron media were mixed in equal quan-
tibies. The final pH was 5.4 as compared with 6.8 of the spor-
ulation medium alone. Aliquots from the liquid sporulation medium
showing good growth of the Streptomyces species were transferred
to the mixed solution. This was done to determine if the anti-
bioticproducers could become gradually adapted to acid iron con-
ditions. The flasks were shaken and aerated as before. After
seven days, no visible growth was noticed in the flasks. How-
ever, filtrates were prepared and tested against the iron bacteria
as before. The filtrates did not inhibit the iron bacteria.
The flasks were allowed to incubate for three weeks, then
retested against iron bacteria. At this time, growth was seen in
the flask containing S. aureofaciens , but no growth was observed
in flasks containing S. antibioticus 1 S. niveus and S. spheroides .
The S. aureofaciens aliquot inhibited Karen iron bacteria for four
days beyond the control tithe, but no other species of iron bac
teria were inhibited. The other antibiotic producers did not inhibit
any strain of iron bacteria.
It had been previous .y reported that Streptornyces did not
grow well in Czapeks medium. However, it was found that growth
did occur after an incubation time of 40 days. Tests conducted
with aliquots from flasks of the various Streptomyces species in
this medium showed that inhibition of iron bacteria was of the
same order of magnitude as those from the original sporulation
medium. Since the Streptomyces antibiotics inhibited iron bac-
teria when transferred from Czapeks solution to synthetic mine
drainage, attempts were made to adapt the Streptomyces species
to coal mine environments. In an attempt at adaption, aliquots
of the various species of Streptomyces, actively growing in Czapeks
nutrient, were transferred to coal and mine drainage in flasks.
Coal (10 g) and pyrite (2 g) were added to each flask containing
100 ml. of synthetic I mine drainage, (Table 1 ). Ferrous sul-
fate was not added to the medium since it was felt that coal and
pyrite would supply the iron and sulfur needed. An additional mod-
ification was made by adding one ml of 10 percent sucrose to
the medium as an added energy source required by the Streptomyces.
The pH of the medium was adjusted to pH 3.9 using sulfuric acid.
The fLasks were aerated and shaken as before. After six days,
growth appeared on the liquid surface of each flask. Aliquots
were transferred to Czapeks agar. In all instances, the
Streptomyces grew on the agar as bright yellow colonies; the S.
spheroides colonies were lytic showing clear areas around each
colony. It is evd.dent that variants have occurred as these col-
onies differ markedly from their normal chalky-white appearance.
The characteristic earthy odor, although not as strong as normal,
98
-------
Table 21 - Inhibitory Effect of Coal-Grown Streptomyces (Sucrose Added)
(16 Days)
In partial oxidation, the lower portion of the liquid is clear, sediment is white
(unoxidized iron), and upper portion is orange (oxidized) iron.
2 Complete oxidation the liquid becomes entirely orange and sediment is entirely orange.
3 In this test, iron became partially oxidized and then cleared at 11 days,
followed by partial oxidation at 18 days.
Inhibitory Effect
Greater with tine
Greater with time
Almost complete with
time
Same as above
Greater with time
Oxidation of Ferrous
IronBacterial Control Tubes
(Days)
Streptomyces
Species
S. spheroidea
Iron
Bacteria
F. sulfo
Degree of Ferrous Iron
Oxidation (Days)
2
complete 2 11 partia]) 22 partial
2
S.
S.
spheroides
spheroides
F. ferro
T. ferro
4
4
partial 22 partial
partial U clear 3 22
partial
2
2
S.
spheroides
Karen
4
partial 17 complete
2
S.
apheroides
Robena
4
complete
2
S.
niveuc
F. sulfo
4
partial 22 partial
2
S.
niveus
F. ferro
4
partial 22 partial
2
S.
niveus
T. ferro
4
partial 11 complete
2
S.
niveus
Karen
4
partial 11 complete
2
S.
niveus
Robena
4
partial 22 partial
2
Not increased with
time, effect is
partial
Slight partial inhib
tion, not increased
with time
Greater with time
Greater with time
Partial inhibition,
not increased with
time
-------
was still present. It is apparent that these Streptomyces
strains have adapted to a new environment.
Aliquots were tested as before against the iron bac-
teria. However, no inhibition occurred with the exception of
S. aureofaciens which inhibited Karen iron bacteria only. It
appears that while the Streptomyces species adapted to acid
mine environments, the ability to produce effective antibiotics
was impaired.
Growth of the Streptomyces in flasks containing coal in
mine drainage was allowed to continue for 16 days, then aliquots
retested against the iron bacteria in liquid synthetic acid mine
drainage. In this group of tests a 1:5 ratio of iron bacteria
to Streptomyces bacteria was used. The Streptomyces aliquots
were increased in order to increase the chances of demonstrating
antibacterial action against the iron bacteria.
The results shown on Table 21 indicate some very interest-
ing phenomena; some of the cultures passed from complete oxidation
to partial or almost complete clearing with time. The tubes which
showed bottom-clearing were marked and it was found that the
bottom cleared area tended to rise upward in all tubes. It is
evident that this marked clearing is due to the growth of the
Streptoinyces species as the liquid assumed a milky, cloudy ap-
pearance caused by bacterial growth. This reversal of the orange
color of oxidized iron had not occurred before. It was originally
thought that the orange color of oxidized iron was irreversible,
since it is a chemical change resulting from the growth of the
iron bacteria. The other two species of antibioticproducers were
not included in this test, as previous results showed them to be
weaker. In summary, larger aliquots of some Streptomyces, grown
in flasks containing coal, sucrose and acid drainage, showed in-
hibitory effects against the iron bacteria. It might be that Fe
III was reduced by pyrite catalytically.
Growth of the Streptomyces species in coal flasks was allowed
to continue for an additional five days for a total of 21 days,
then aliquots retested against iron bacteria at a 1:1 ratio
The results are shown in Table 22. It can be seen that S. niveus
and S. antibioticus show fairly good inhibition of the iron bac-
teria. It is evident from the results that the Streptomyces
require a good deal of time to become adapted to new growth con-
ditions as no inhibition was shown in the early stages of growth
(six days); but fairly good inhibition was evident at 21 days.
Increasing the ratio of Streptomyces growth aliquot over the iron
bacteria also showed a good inhibitory effect when Streptomyces
cultures were grown for 16 days.
Results indicated that some types of Streptomyces partially
100
-------
Table 22- Inhibitory Effect of Coal-Grown Streptomyces (Sucrose Added)
(21 Days)
St re ptomyce S
Species
S. spheroides
S. spheroides
S. spheroides
S. spheroides
S. apheroides
Iron
Bacteria
F. sulfo
F. ferro
T. ferro
Karen
Robena
Degree of Ferrous Iron
Oxidation (Days)
3 partial 1 6 compLete 2
a
N
,,
a
Oxidation of Ferrous
IronBacterial Control Tubes
(Days)
2
2
2
2
2
Inhibitory Effect
Low inhibitory effect
U N N
a a
a
U N
N
U
S. niveus
S. niveus
S. rtiVeUs
S. niveus
S. niveus
F. sulfo
F. ferro
T. ferro
Karen
Robena
2 partial 14 complete
a N
a a
N N
2
2
2
1 Partial oxidation the liquid portion is partially clear, and sediment is white (unoxidized iron).
2
2
S.
antibioticus
F. sulfo
10 partial
16 complete
2
S.
antibioticus
F. ferro
8 partial
14 complete
2
S.
antibioticus
T. ferro
6 partial
14 complete
2
S.
antibioticus
Karen
6 partial
16 complete
2
S.
antibioticus
Robena
8 partial
14 complete
2
S.
aureofaciens
F. sulfo
2
partial
3 complete
2
S.
aureofaciens
F. ferro
2
partial
3 complete
2
S.
aureofaciens
T. ferro
2
partial
6 complete
2
S.
aureofaciens
Karen
2
partial 6 complete
2
S.
aureofaciens
Robena
2
partial
3 complete
2
Partial inhibition
a a
a
I. a
N N
Partial inhibition
a N
a i v
N
a a
Low
a
U
I .
N
inhibitory effect
iv N
U i i
$ N
2 Complete oxidation the liquid becomes entirely orange and sediment is entirely orange.
-------
inhibited iron bacteria for 22 days in liquid synthetic acid
mine drainage. The streptomyces were grown in flasks contain-
ing coal, pyrite and synthetic mine drainage with sucrose added
as an additional supplement for growth. The inhibitory action
was clearly shown when each antibioticproducer was tested sep-
arately against each strain of iron bapteria in synthetic acid
mine drainage without additional supplements. Test tubes showed
an initial bottom clearing which gradually increased with time
until 40 days when tests were terminated. The positive bac-
terial controls became completely oxidized in two days and re-
mained oxidized until tests were stopped. Negative control tubes
remained clear throughout the test. The tubes containing Strepto-
myces and iron bacteria cleared at the bottom first, then grad-
ually the clearing of the orange color of oxidized iron was re-
placed by a milky, cloudy appearance. The ti be contents became
clear of oxidized iron except for iron which had adhered to the
glass above the liquid surfaces. Color photographs were taken
when tests were terminated and sent to certain representatives of
our sponsors in this program. In addition, pH tests done at
this time showed the inhibited tubes averag about 2.8 as com-
pared with 2.5 of positive bacterial control% tubes. This in-
dicated that iron was reduced and was in some way connected with
the interaction of Streptomyces and/or antibiotic products with
the iron bacteria. Tests conducted at a later time to help
clarify these points are described below.
In order to determine if the inhibited tubes contained
actively growing Streptomyces, aliquots were aseptically removed
and plated on Czapeks agar. After- incubation, yellow colonies
of the adapted Streptomyces types were subsequently isolated.
Tests were also made, to be described in a later part of this
report, showing that iron bacteria do not grow on Czapeks agar
nor do Streptomyces oxidize iron.
In summary, results indicate that certain types of Strep-
tomyces will partially adapt to acid mine drainage if some sucrose
is present. These adapted straiiis inhibit iron bacteria in acid
mine drainage for varying periods of time. Methods of obtaining
full adaptation without sucrose are described below.
Full Adaptation of Streptomyces
Studies Were made to determine if the various strains of
Streptomyces could fully adapt to acid/iron conditions, and con-
tinue to produce effective antibiotics against the iron bacteria.
Aliquots from the four strains of Streptomyces, growing in coal,
pyrite,. sucrose and mine drainage, were transferred to fresh ster-
ilized flasks of coal, pyrite and acid mine drainage without
sucrose. These flasks, plain and irradiated, were aerated and
shaken. At various time intervals, aliquots were removed and
102
-------
tested against all strains of iron bacteria in tubes of synthetic
mine drainage. Aliquots tested after seven days of growth were
negative for inhibitory powers.
Growth of the Streptomyces was allowed to continue for
an additional ten days, or a total time of 17 days. Aliquots
were then tested against iron bacteria at a 5:1 Streptomyces/iron
bacteria ratio. Streptomyces aliquots were removed and additional
tests made after total incubation times of 25 and 35 days at the
same ratio. The results are shown on Tables 23, 24, and 25. It
can be seen that only S. aureofaciens inhibited the iron bacteria
regardless of the age 3 f the Streptomyces culture. The S. spheroides ,
S. niveus and S. antibioticus had previously shown inhiDitory ac-
tion when grown in pyrite, coal, drainage and sucrose. When ad-
apted to an autotrophic environment of coal, pyrite and acid
drainage, the inhibitory action of these three species appeared
to have been lost. Aliquots from Streptomyces coal flasks were
placed onto Czapeks agar and incubated. The yellow colonies of
adapted Streptomyces were isolated from all flasks. Therefore,
the various Streptomyces strains have adapted to, or survived in
the coal environment a evidenced by the growth on plates. How-
ever, some of these species lost their ability to produce effective
antibiotics against the iron bacteria.
In summary, it appears that S. aureofaciens fully adapted
to an acid coal environment and produced antibiotics effective
against the iron bacteria at a 5:1 Streptomyces iron bacteria ratio.
The other Streptomyces species will grow under full acid/iron
conditions without supplements, but appear to have lost other anti-
biotic powers. It remains to be seen what effect the antibiotic
producers would have in coal pile containers, especially on acid
production and on iron oxidation.
Small flask cultures (200 ml) of S. aureofaciens and S.
spheroides were used to seed 5 liter quantities of synthetic acid
mine drainage containing coal and acidified pyrite. Culturing
was done in large batch quantities to determine if better growth
of the Streptomyces would occur. The nutrient materials were
added to sterile, 2 gallon containers fitted with sterile qlass
fritted bubblers. Air intake and outlet lines were fitted with
sterile millipore air filters
The seed cultures were added, air supply started and
the containers shaken continuously. After 6 days of incubation
excellent growth of the two antibiotic-producing Streptomyces
speceis were obtained. These were then used to test their ef-
fects on flooded upwardflowing vertical acidproducing systems
as described elsewhere.
103
-------
Tabic 23 - Inhibitory Effect of Coal-Grown Streptomyces
(No Addi ti yes) 1 7 Days
Partial oxidation The liquid portion is partially clear, and sediment is
white ferrous iron.
Complete oxidation The liquid becomes entirely orange and sediment is
entirely orange.
Control tubes containing no Streptomyces were completely oxidized in two days.
H
0
S.
S.
S.
S.
Streptomyces
Species
Iron
Bacteria
F. sulfo
F. ferro
T. ferro
Karen
Robena
Degree
of Ferrous IronW
Oxidation (Days)
2
2
2
2
2
complete
complete
complete
complete
complete
S. apheroidsi
S. epheroides
S. apheroides
S. apheroides
S. spheroides
S. niveus
S. niveus
Sd niveus
S. niveus
S. niveus
F. sulfo
F. ferro
T. ferro
Karen
Robena
2
2
2
2
2
complete
complete
complete
complete
complete
antibioticus
antibibticus
antibioticus
antibioticus
antibioticus
F. sulfo
F. ferro
T. ferro
Karen
Robena
2
2
2
2
2
complete
complete
complete
complete
complete
S. aureofaciens
S. aureofaciens
S. aureofaciens
S. aureofaciens
S. aureofaciens
F. sulfo
F. ferro
T. ferro
Karen
Robena
3
3
3
6
6
partial
partial
partial
complete
partial
17
17
17
17
Inhibitory Effect
None
I ,
ft
None
ft
ft
It
ft
None
I,
It
Partial inhibition
It It
ft I ,
I
It
complete
complete
complete
complete
-------
Table 24 - Inhibitory Effect of Coal-Grown Streptomyces
(No Additiv c 25 Days
Streptornyces
Species
S. spheroides
S. spheroides
S. spheroides
S. spheroides
S. spheroides
Iron
Bacteria
F. sulfo
F. ferro
T. ferro
Karen
Rob en a
Degree of Ferrous Iron 1
- Oxidation (Days)
2 complete
2 complete
2 complete
6 complete
2 complete
Inhibitory Effect
None
ft
I
ft
S. niveus
S. niveus
S. niveus
S. niveus
S. niveus
S. antibioticus
S. antibioticus
S. antibioticus
S. antibioticus
S. antibioticus
S aureofaciens
S. aureofaciens
S. aureofaciens
S. aureofaciens
S. aureofaciens
F. sulfo
F. ferro
T. ferro
Karen
Robena
F. sulfo
F. ferro
T. ferro
Karen
Roben a
F. sulfo
F. ferro
T. ferro
Karen
Robena
2 complete
2 complete
2 complete
2 complete
2 complete
2 complete
2 complete
2 complete
6 complete
2 complete
6 partial
6 partial
6 partial
6 complete
6 partial
partial (2)
partia l( 2 )
partial (2)
9 partial 2
None
It
I
Partial inhibition
II II
Partial inhibition
It It
It U
Low inhibitory effect
Partial inhibiti n
( 1 Partial oxidation The liquid portion is partially clear, and sediment is
white (unoxidized iron).
Complete oxidation The liquid becomes entirely orange and sediment is
entirely orange.
Control tubes containing no Streptomyces were completely oxidized in two days.
2 ThiS partial inhibition appeared to be stable at this point when chemistries
were done.
0
01
N one
I
I,
I
I
9
9
9
-------
Table 25 - Inhibitory Effect of Coal-Grown Streptomyces
(Nn Add I t i v e si - 35 J a s ____
Streptoinyces
Species
I ron
Bacteria
Degree of Ferrous Iron( 1 )
Oxidation (Days)
Inhibitory Effect
S. spheroides
S. spheroidee
S. spheroides
S. spheroides
S. apheroides
F. stilfo
F. ferro
T. ferro
Karen
Robena
2 complete
2 complete
2 complete
2 complete
2 complete
None
I ,
ft
ft
ft
S. niveus
S. niveus
S. niveus
0 S. niveus
S. niveus
F. sulfo
F. ferro
T, ferro
Karen
Robena
2 complete
2 complete
2 complete
2 complete
2 complete
None
I,
H
I
I
S. antibioticus
S. antibioticus
S. antibioticus
S. antibioticus
S. antibioticus
F. sulfo
F. ferro
T. ferro
Karen
Robe n a
2 complete
2 complete
2 complete
2 complete
2 complete
None
I,
I
Si
I
Partial inhibition
5, 5,
I, 55
5, 5,
Si SI
1 Partial oxidation - The
white (unoxidized iron).
Complete oxidation - The
entirely orange.
Control tubes containing
liquid portion is partially clear, and sediment is
liquid becomes entirely orange and sediment is
S.
S.
aureofaciens
aureofaciens
F. sulfo
F. ferro
4
4
complete6
complete 6
partial-lO
partia]. lO
partial
partial
S.
aureofaciens
T, ferro
4
coxnplete6
partial lO
partial
S.
aureofaciens
Karen
4
complete6
partial lO
partial
S.
aure.ofaciens
Robena
4
complete6
partiallU
partial
no treptomyces were completely oxidized in two days.
-------
Tests to Determine if Streptomyces Oxidized Iron
Streptomyces colonies from all stages of partial and
fully adapted growth were isolated on Czapeks agar, then picked
and inoculated into test tubes containing synthetic acid mine
drainage. After incubation, tube contents became cloudy with
growth, but iron was never oxidized. These yellow colonies of
adapted Streptomyces will not oxidize iron. In addition, all
strains of iron bacteria were placed on Czapeks agar to determine
if they would grow and be mistaken for Streptomyces. It was
found that iron bacteria would not grow on Czapeks agar.
Chemical Analyses of Streptomyces-Inhibited Tubes
It was shown previously that some Streptomyces species
partially blocked ferrous iron oxidation in test tube studies.
It was observed that some Streptomyces species cleared oxidized
iron in nutrient synthetic acid-mine drainage in test tubes.
In some cases, the iron bacteria oxidized iron, but this was fol-
lowed by subsequent clearing from the bottom of the tubes upr
ward. This clearing was caused by the action of the Streptomyces
(or byproducts). It was not known if a chemical or antibiotic
produced by the Streptomyces species actually reduced iron.
In order to better define the chemical nature of partial
iron bacteria inhibition, the Streptomyces and iron bacteria tubes
were analyzed after 9 days of partial inhibition. These were com-
pared with a positive iron bacterial control tube. The results
are shown on Table 26. It can be readily seen that the ratio
of ferrous to ferric iron has been changed. The tube containing
S. aureofaciens and Robena iron bacteria shored that about 55 per-
cent of the total iron was found in the ferrous state. The bac-
terial control tube shows only about 2% of the iron in the ferrous
state. The other analytical results showed very little change.
Testing Streptomyces for Presence of IronReducing Substances
A series of tests was set up to determine if the Strepto-
myces produced an ironreducing substance. A ferric sulfate
solution was prepared, sterilized by filtration and added to nutrient
synthetic acid mine drainage in the same quantity as ferrous iron
is added to prepare the synthetic acid iron solution normally used
to test for the presence of iron oxidation. Test tubes were set
up consisting of five series of 3 tests each. Ten ml oE acid
ferric iron solution was added to 1 tube of each series; a 0.5
ml quantity of S. niveus was added to the second tube of each
series. A 0.5 ml quantity of nonoxidized synthetic ferrous iron
solution was added to the third control tube of each series to
compensate for the addition of 0.5 ml quantities of Streptomyces
grown in synthetic mine drainage containing coal. One additionaltube
107
-------
Sample Designation ____
S. aureofaciens Vs F. sulfo
S aureofaciena VS F. ferro
0
03
S. aureofaciene vs T. ferro
S. aureofaciens vs Robena 208
Pos. Control Robena
Table 26
- Analyses of s treptomyces -Inhibited Tubes
Total Iron Sulfates
ppm ppm p xn ppm
118 208 326 1650 2.52
5 385 390 1630 2.80
32 333 365 1425 2.90
169 377 1850 2.95
372 380 1400 2.80
Total Acid
ppm
1025
1018
1025
1038
1062
8
-------
was set up containing the ferric iron solution with nothing else
added. Each set of three tubes was analyzed at one time with each
series being analyzed one week apart. Each set was discarded after
analysis to avoid contamination. The final pH of the ferric
acid iron solution was 2.5 which is lower than the normally used
ferrous acid iron solution (3.6).
The results shown in Table 27 indicate little change in the
chemical analyses, except for unexplained variation in sulfates.
It is evident that an ironreducing agent is not present under
these test conditions. There was no indication that the Strepto
myces species were growing in the ferric iron synthetic drainage
media. It appears that the clearing caused by Streptomyces is
possibly some interaction of Streptomyces and iron bacteria on
ferric iron.
Microscopic Studies of Adapted Streptomyces
A study was made on S. aureofaciens at various stages of
adaption to roughly determine if any changes occurred during
adaption. This was done by placing small aliquots on glass slides,
staining, and observing the structure in the microscope. All
slides were compared with the original culture obtained from the
American Type Culture Collection. The appearance of the Strepto-
myces was the same with coaladapted Streptomyces, the yellow col-
onies on Czapeks agar taken from coal adapted Streptomyces and
from Strpetomyces growing in a tube of liquid synthetic mine drain-
age. The appearance of each of these samples was the same as the
original stock culture. The colonies on plates differed as the
original stock culture growing on Czapeks agar had a white pow-
dery appearance, while adapted coal colonies were yellow on Czapeks
agar. However, the microscopic appearance of all cultures were
the same. Thus, we can be fairly certain thea the S. aureofaciens
has adapted to a coal environment. Further testing in flowing
streams through coal piles made to determine if the various
Streptomyces strains will continue to produce antibiotics effective
against the iron and sulfur bacteria is described below.
S. aureofaciens appears to be a good candidate for an in-
hibitor as it produces chiorotetracyclene, an amphoteric antibiotic
compound continaing both nitrogen and non-ionic chlorine active
against various bacteria. It also produces tetracycline in a
chlorinepoor environment.
Streptomyces in Flooded Upward Flowing Vertical Systems
Two of three containers with Robena bacteria producing acid
were each inoculated with 100 ml of Streptomyces aureofaciens cul-
tures and two of another group of three were inoculated with the
same volume of S. spheroides . The effluents were monitored regular-
109
-------
Table 27 - Testing for Presence of Streptomyces-Induced Iron Reduction
Time of Fe Fe 4 Total Acidity Sulfates
Sample analyses ppm ppm iron-ppm _ ppm ppm pH
Fe in Syn I Immediate 11(1) 810(2) 810 2863 3875 2.25
S. spheroides Immediate 10 675 675 1863 2125 2.45
S. niveus Immediate 9 622 622 1869 2900 2,50
Control Immediate 39 640 670 1856 2250 2,45
S. apheroides 1 week 14 650 650 2113 3125 2.45
S. niveus 1 week 14 655 655 1969 3500 2.50
Control 1 week 41 640 705 2019 3550 2.50
S. spheroides 2nd week 10 610 610 2083 2750 2,30
o S. niveus 2nd week 12 628 628 2125 2250 2.35
Control 2nd week 34 645 665 2125 2250 2,32
S. spheroides 3rd week 12 618 618 2225 4200 2,25
S. niveus 3rd week 14 618 618 2275 4200 2,30
Control 3rd week 38 640 675 2233 4000 2.35
S. spheroides 4th week 15 590 590 2200 3125 2.15
S. niveus 4th week 18 600 600 2275 3250 2,25
Control 4th week 40 600 630 2250 3500 2.1.5
(1) This number is believed to be blank due to absorption of ferric
sulfate color at this wave length.
(2) These numbers are approximate since low reading obtained from
ferrous iron were blank.
-------
ly for pH and total cidity. Results are shown in Tables 28 through
30 and in Figures 33 through 35.
Table 28 - Viability of Robena Bacteria in Systems Inoculated with
S. Aureofaciens
Cells per Milliliter
Days After #1 #2 #3
Inoculation Control Test Test
0 1,000 300,000 2,000
7 9,000 8,300 2,300
14 4,000 1,000 100
21 4,000 400 1,000
Table 29 - Viability of Robena Bacteria in Systems Inoculated with
S. Spheroides
Cells per Milliliter
Days After #1 #2 #3
Inoculation Control Test Test
0 3,000 9,000 7,000
7 14,000 1,100 100
14 700 12,000 2,300
21 1,000 2,500 1,400
It can be seen that S. aureofaciens has some inhibitory
effect on both the chemistry and microbiology of the systems but
that S. spheroides has no such effect. Viabilities in systems
with . aureofaciens fell off significantly, acid production was
decre sed and the mean pH of the two test systems along with data
on other systems generally showed a rise. A statistical analysis
of these data along with data on other systems is given in Ap-
pendix 1
111
-------
Table 30 - Test Data on Streptomyces as Inhibitor
Days After
Adding
S. spherofdes
No. 4
Control
Acidity
No.
Test
(ppm)
5 No. 6
Test
t.J
NO. 4
Control
2.80
2.80
2.92
2.90
2.90
2.90
2.90
2.90
2.80
2.80
3.00
2.80
pH
No. 5
Test
2.95
2.95
3.05
2.92
2.70
2.60
2.70
2.70
3.10
2.90
2.90
3.00
2.90
No. b
Test
2.89
3.10
3.10
2.90
2.80
270
2.80
2.60
2.90
2.70
2.90
2.90
2.80
320
640
520
360
500
350
210
400
480
420
1050
550
320
212 610
370 280
380 380
420 580
400 380
770 400
890 510
820 480
760 450
620 450
390 390
150 140
350 410
Oxidation Times
(days to cloudinq)
Vi abilities
( thousands of cells/mi)
-3
5
11
17
25
32
39
46
53
60
74
81
-1
7
14
21
28
35
42
49
56
63
70
3
14
0.7
1.0
1.6
74
2.8
5
<0.1
10
--
9
Li
12
2.5
1.1
4.8
0.8
0.7
<0.1
0.1
--
7
<0.1
2.3
1.4
0.8
3.7
0.1
4
<0.1
0.9
--
---
4
3
4
4
3
3
3
6
3
4
--
4
3
4
3
3
3
3
8
4
5
3
5
3
3
3
3
3.
8
4
4
-------
I I I I I I I I I I
p 6 t3Test 0
-
0
-I
U 4
0
I_a 4J _____
-.- - - -.-
UI #1 Control
>1 2_
0
I i i I I i I i 1 i I i I I I i I t 1 t I i I i I i I i I
.0
12 16 20 24 28 32 36 40 44 48 52 56 60 64 68
Days After Adding S. aureofaciens
Figure 33 - Effect of S. aureofaciens on Oxidation Times of Robena Bacteria
-------
- 03 Tast -
12 14 20 24 28 32 36 40 44 48 S2 56 60 64 68
0 4
Days After First Addition of Inhibitor
72
Figure ,4 - Effect of S. aureofaciens on pH of Robena Cultures in
Upward-flowing Vertical Streams
Figure 35 - Effect of S. aureofaciens on Acid Production by
Robena Bacteria on Upward-flowing Vertical Streams
t
0 .
20 24 28 32 36 40 44 40 52 56 60 64 61 72
Days After First Addition of Inhibitor
114
-------
SECTION 14
ACKNOWLEDGEMENTS
Dr. Robert P. Zimmerer performed services of electron micro-
scopy and consultant services on overall conduct of the project.
Dr. Vincent Gerenscer was consulted on the characteristics of
Caulobacters and he furnished four stains for evaluation. This
project was greatly added by supportof Dr. David R. Maneval,
Director of Research and Development for the Pennsylvania Depart
ment of Mines and Mineral Industries; of Dr. H. B. Charmbury,
Secretary of this department; of Mr. Rc a1d D. Hill, Chief of Mine
Drainage Pollution Control Activities o the Federal Water Pollution
Control Administration.
The contributions of the United States Steel Corporation was
of great value to the project, and are gratefully acknowledged.
115
-------
SECTION 15
REFERENCES
Adams, Mark U., Bacteriophages , Interscience Publishers, Inc.,
New York, 1959
Alexander, N., Introduction to Soil Microbiolη gy , John Wiley
and Sons, Inc., 1967
American Public Health Association, Standard Methods for the
Examination of Water and Wastewater. , 12th Edition, 1965
Bradley, D.E.., Ultrastructure of Bacteriophages an Bacter-
iocins, Bacteriological Reveiws 33: 230314, 1967
Bradley, D.E., The Isolation and Morphology of Some New Bac-
teriophages Specific for Bacillus and Acetobacter Species,
J. Gen. Microbiology 41: 233241, 1965
Brownlee, K.A., Industrial Experimentation , Chemical Publish-
ing Company, New York,1953
Brownlee, K.A., Statistical Theory and Methodology in Science
and Engineering , John Wiley and Sons, New York 1965
Chruchill, Arthur V. and Leathen, William W., Development of
Microbiological Sludge Inhibitors, ASO Technical Report 61-193
September 1962
Gerencser, V. ,Personal Communication, May, 1969.
Hach Chemical Company; Ames, Iowa
Hesseltire, C.W., Benedict, R.G., and Pridham, T.G., Useful
Criteria for Species Differentiation in the Genus Streptomyces,
Annals of the New York Academy of Sciences , 60, Art. 1 pp 136-151,
1954.
Poindexter, J.S., Biological Properties and Classification of
the Caulobacter Group, Bacteriological Reveiws 28: 231-295,
1964
Shearer, R.E. and Everson, W.A., Elimination of Pollution in Mine
Drainage, P.R. No. 2 (Jan-June) Project No. CR-54 , Commonwealth
of Pennsylvania, Department of Mines and Mineral Industries,
Shearer, R.E. and Everson, W.A., Elimination of Pollution in Mine
Drainage, R.R. No. 6 (Jan- June) Project No. CR-62 , Commonwealth
of Pennsylvania, Department of Mines and Mineral Industries, 1967
117
-------
REFERENCES (CONTINUED)
Shearer, R.E. et al Second Symposium on Acid Mine Drainage ,
Coal Industry Advisory Committee, 1968
Stanier, R.Y., Doudoroff, N., Adelberg, E.A., The Microbiological
World , Prentice-Hall, Inc., New York 1961
T ie American Type Culture Collection, Catalogue of Strains .
Eighth Edition, 1968.
Waksman, S.A., The Actinomycetes , Ronald Press Company, New
York, 1967.
Zimrnerer, R.P. - Personal Communication , 1969
118
-------
SECTION 16
GLOSSARY
Adaption - Any change in an organism which increases its f it-
ness to the environment.
Autocatalytic Inhibitor - An inhibitor produced in the death
phase of microorganisms which when applied to fresh viable
microorganisms causes them to produce more of the inhibitor
and induces death.
Autotrophic Needing only inorganic compounds for nutrition.
Bacterial Reduction - A decrease in bacterial numbers.
Bactericide - An agent which kills bacteria.
Bacteriocin - A natural class of highly specific antibiotics.
Bacteriophage - Antonomous microbes analogous to plant and
animal viruses but obligately parasitic on bacteria.
Bacteriostatic - Prevention of the growth of bacteria without
destruction.
Caulobacters Grainnegative, unicellular, stalked bacteria
which multiply by binary fission. During reproduction both
stalked and non-stalked forms exist.
Flagella - Whip-like appendages used by bacteria singly or in
number for motility.
Filtrate Inhibitor - Inhibitor obtained by inoculating uncon-
centrated natural inhibitor into a nutrient medium, along with
iron bacteria. After incubating, the material is millipore-
filtered to isolate the filtrate.
Inhibitor - An agent which slows or interferes with growth
of bacteria.
Lysis - Dissolution of the living cells of bacteria.
Mutation A sudden variation (change) in microorganisms. A
transmissible variation that is likely to be permanent, seemingly
arising suddenly and spontaneously.
119
-------
GLOSSARY (CONTINUED)
Oxidation Times - The time (in days) required for bacteria to
fully oxidize ferrous iron (as part of synthetic mine drainage).
This gives some measure of bacterial inhibition as test quan-
tities of iron bacteria and inhibitor are always compared with
a control tube of ferrous iron mine drainage containing the
same quantity of iron bacteria as the test. A negative control
tube is also used with each set of tests in which the ferrous
iron should remain unoxidized for long periods of time.
Plaques Zones of lysis.
Potential Phage Sources - Natural environmental materials such
as soil, water, refuse, etc. which might yield quantities of
phage upon treatment with the proper bacteria.
Satellite Bacteria - A few sparsely located resistant bacteria
appearing in an otherwise clear zone of an antibiotic disk
where sensitive bacteria have been lysed.
Seed Culture - A fairly large culture of bacteria growing in
a nutrient and aliquots of which are used to inoculate other
cultures or systems.
Streptomyces - Members of a genus of the order Actinomycetales.
Members of this genus produce most of the antibiotics and are
most often soil microorganisms.
Variant - Similar to mutant.
Viable Able to reproduce themselves.
Viability Count - Numbers of living cells per milliliter of
liquid.
Viruses Infectious filterable particles (usually less than
300 mi].limicron in diameter) that can reproduce only within
a specific host cell.
120
-------
APPENDIX 1
Statistical Analysis of Inhibition in Various Streams
In order to assess the confidence that inhibition
of iron/sulfur bacteria had been obtained in different
experiments, a statistical program was set up on a General
Electric #400 Time Sharing Programming System. The arith-
metic mean and standard deviation of various sets of data
were obtained from statistical tables. The results together
with the underlying data are given in Table 31 .
The data selected for the calculations arbitrar-
ily on what appeared to show possible differences. They
covered effluents from upwardflowing streams through flood-
ed coal piles inhibited by natural waters and by cultures
of Caulobacters and of Streptomyces.
Of 19 sets of data evaluated, only two fell below
the 95% confidence level considered necessary for reasonable
assurance that there are true differences and that inhibition
was attained. One of these sets was for effect on pH of
raw natural inhibitor on acidproducing bacteria from Karen
mine effluents. To counteract this questionable level ( 90%),
the effect on both sulfate and total iron production in the
same test had a 99.5% confidence level. The other set of
data falling short of good confidence was that on effect
of one of two tests of S. aureofaciens on pH. The comparison
set showed a 99.5% confidence level , and the set on the
effect of S. aureofaciens on acid concentration showed a
confidence level of 99.5%.
Considerable variations are noted in the time for
onset of inhibition and in duration of inhibition. These
variations on time are possibly due to variations in re-
sistance of various strains of acidproducing bacteria.
Decline in acidity and iron content of the control streams
are frequently noted, and this is possibly due to depletion
of nutrient pyrite in the coal. Because of limited previous
experience, it was not possible to predict in advance when
additional inhibition had to be added. This may account for
limited periods of inhibition shown in the data.
121
-------
Table 3]. - Statistical Data on Characteristics of Inhibited Streams
p .,
Effluent
Number
Type of Inhibitor
Stream
Characteristic Value before
Tested Inhibition
ppm acid 90
N 100
N
Interval
Tested
(Days after
Adding
Inhibitorj
69231
Number
of
Values
25
25
25
Mean
Yalue
908.8
322.08
80
Standard
Deviation
858.3
320.44
74.3
Students
NT Value
:
3.20
4.81
I
2
3
Control for 23
Raw Natural
Raw Filtrate
1
2
3
Control for 2,3
Raw Natural
Raw Filtrate
ppm 504 1000
N 975
N 800
6188
28
27
27
852.5
481
311.1
526.8
349
157
3.07
5.12
4
S
6
Control for 5,6
Raw Natural
Raw Filtrate
pH 3.6
N 4.1
N 4.2
35169
N
23
23
23
4.0
5.0
4.8
1.22
1.17
1.11
2.84
2.33
1
2
3
Control for 2,3
Raw Natural
Raw Filtrate
Total iron 7.8
. 32
19
76216
N
21
21
21
252.5
51.3
12.3
263
73.9
17.7
3.37
4.17
4
S
6
Control for 5,6
Raw Natural
Raw Filtrate
Total iron 7
N 14
N 24
56189
N
20
21
21
165.9
21.31
54
253.6
27.9
96.4
-
2.60
1.89
8
9
Caulobacter
Control for 8
Total iron 579
- 517
21114
N
14
14
26.36
160.9
33.89
72.8
6.27
4
S
6
Control for. 5,6
Raw Natural
Raw Filtrate
ppm acid 160
N 106
120
84189
16
16
16
653.4
143.7
220.6
627.1
175.3
301.2
3.12
2.49
10
11
Control for 11
Caulobacter
pM 2.3
N 2.45
41160
17
17
3.69
6.0
.565
.916
8.85
10
11
Control for 11
Caulobacter
ppm acid 5800
4360
41160
18
18
208.1
18.4
111
40.1
6.82
la
2a
3a
Control for 2*, 3a
S. aureofacien.
S. aureofaciens
pH 2.79
N . 2.79
N 3.05
581
N
12
12
12
2.85
2.94
3.46
.214
.156
.294
1.18
5.81
1*
2a
Control for 2a, 3a
S. aureofaciens
ppm acid 850
730
581
12
12
696.7
324.2
253.5
96.05
-
4.78
Confidence
Level
on Difference
from Control, S
.99 5
?99.5
>99 5
>99 5.
>99.5
>97.5
>99 5
>99.5
99
7 .95
>99.5
799 5
99
>99.5
>99.5
>80, <90
p99.5
799.5
-------
APPENDIX 2
Analytical Data on Horizontal Flooded
and Vertical Spray Systems
Tables 32 through 42 contain detailed analytical
data on attempted inhibition in streams through flooded
horizontal systems and downward through vertical unflooded
spray systems with natural inhibitor. In column headings
with numbering such as 1-1, 1-2, 1-3, etc. the first digit
represents the system number. In horizontal systems, the
second digit represents the sample port starting at the
influent end. Thus 2-4 represents the fourth sample port
in system number two. In the vertical systems, the second
digit represents the sample port number starting at the top.
Thus, 2-4 again represents the fourth port in system number
two.
For purposes of economy, not all ports were
sampled every time and data are absent as represented by
dashes. Some data are also absent because of inability to
obtain samples because of some mechanical difficulty.
No evidence of inhibition was seen in the data.
Some anomalous values appearing in the tables are due to
alkalinity of pyrite added as nutrient to replace depleted
pyrite.
123
-------
Date and
Days After
Adding
Inhibitor
11/11
11/21 9
11/26 14
65 65 385 780 1040 370
150 340 865 1355 1974 1150
110 35 70 170 240 115
1/8 57 250 985 -- 2325 3175 2910
1/14 63 185 210 - 1615 2855 4525
1/21 70 140 420 1485 2460 5425
1/28 17 135 445 - 1365 2270 6230
35 105 -- 320 565 5405
20 40 --- 135 275 4580
70 205 - 475 415 940
15 35 - 360 605 132
3/5 0 30 100 -- 240 540 1190
3/11 6 45 235 - 545 1285 1240
3/18 13 20 115 245 575 - 910
3/25 20 40 190 - 340 695 1045
27 50 120 - 205 515 780
34 30 260 - 185 430 630
41 40 110 . 125 340 420
48 50 160 -- 285 590 885
55 60 260 -- 520 815 1145
5/6 62 30 180 . 130 330 460
5/14 70 100 115 - 105 255 325
5/20 76 46 130 --- 140 310 400
5/26 82 26 102 -- 94 265 360
5 15 10 195 485 785
20 85 210 235 580 1415
10 10 120 140 405 1335
-15 120 --- 290 605 1630
5 105 205 SOS 1635
25 60 -- 145 285 860
10 70 -- 180 300 1035
25 55 - 145 320 890
20 35 - 45 120 420
25 125 15 165 565
20 60 -- 10 285 815
20 50 130 250 . 720
100 65 - 50 320 905
30 105 320 545 1385
40 310 -- 430 625 1695
20 155 --- 390 605 1530
25 105 345 550 1615
20 250 --- 110 490 1050
25 335 380 420 1040
45 255 255 485 1230
35 215 --- 160 365 720
30 230 80 300 755
14 410 235 300 1010
4 295 - 305 435 855
12 150 230 420 700
10 1 13 14 450
Table 32 - Total Acidity (ppm CaCO 3 equiv.)
Along Horizontal System
12/9 27
12/16 34
12/25 44
Robena bacteria natural inhibitor Robena bacteria -
11 1.2 13 14 1-5 16 17 1-8 19 21 22 2-3 24 2-5 2-6 27 28 2-9
220 900 110 360 2200 160
75 120 115 3055 490 570
35 925 140 245 965 1045
I-
2/5 85
2/11 91
2/18 98
2/26 106
4/1
4/8
4/15
4/22
4/29
6/3 90 19 171 -- 178
6/12 85 68 18 - B
36 550
11 385
-------
Date and
Days After
Adding
Inhibitor
fl/li l
11/21 9
11/26 14
94 350 395 598 760 43
18 44 48 79 105 98
38 29 141 290 276 228
16 29 155 325 580 600
30 350 335 343 590 398
1 17 11 9 41 210
0 3.6 34 50 19 279
0.3 6.2 12.0 8.8 71 490
3/5 0 0 0
3/11 6 2.6 4.2
3/18 13 0.3 11
3/25 20 1.6 10
5/6 62 3 33
5/14 70 16 14
5/20 76 4 10
5/26 82 1 12
36 93 214
48 160 300
36 88 179
43 107 182
11 53
8 35
13 50
8 42
10 51 114
6 35 64
3.6 1
7.3 6.2
5.2 12.2
o 39
1.8 16
0.1 10
0.2 36
2 56
2 42 - -
83 26 31
50 1 40
62 2 84
66 0.5 55
8.3 41 18
28 31 183
47 97 271
60 144 337
61 104 305
53 91 353
24 90 228
67 68 240
48 97 323
26 64 153
9 57 164
40 53 257
63 70 190
73
78
12/3 21
12/9 27
12/16 34
12/26 44
Robena bacterial natural inhibitor Robena bacteria
11 1.2 13 1-4 1.5 16 17 18 19 21 22 23 24 25 26 27 2-8 29
45 200 25 125 470 5
10.4 36.4 17.7 192 177 132
2.6 117 1.0 50 250 145
U,
1/8 57 95 224 --- 650 605 715
1/14 63 15 29 390 815 940
1/21 70 12.5 63 327 518 618
1/28 77 5.7 65 .- 295 460 658
2/5 85
2/11 91
2/18 98
2/26 106
23 6
0 0
0.1 3.1
0.3 0.3
48 83 645
11 31 630
1.8 0.1 45
66 110 254
0 23 59
0.2 23
0.1 5.2
0 4.4
4.7 6.8
0 0
0 0.3
3.1 4.4
4/1
4/8
4/15
4/22
4/29
142 525
54 114 433
12 38.5 150
27 47 204
20 45 158
1 11 55
0.1 0.1 0.4
56 76 269
27 1.6 8 24 71 139
34 0 33 -- 22 53 1G3
41 1.1 8 --- 14 47 67
48 3.4 18 . 41 113 150
55 12 47 . 117 63 307
6/3 90 4 30
6/12 99 05 2
0 21 - 37 83
0 2 - 7 14
Table 33 - Total Iron Along Horizontal System (ppm)
-------
Date and
Days After
Adding
Inhibi tor
Fl /il 1
11/21 9
11/26 14
12/3 21
12/9 27
12/16 34
12/26 44
1/8 57
1/14 63
1/21 70
1/28 77
2/5 85
2/11 91
2/18 98
2/26 106
3/5 0
3/11 6
3/18 13
3/25 20
4/1 27
4/8 34
4/15 41
4/22 48
4/29 55
30 62
0 3
0 43
20 109 115 167 326
o 0 -- 3.5 fl 42
0.5 29 - 12 43 222
0 19 .- 5.5 29 143
0 0 2 8.5 98 0
0 0 --- 0 0 70 0
0 1.0.-- 1.8 0 3.3 0
0 0 --- 43 41 46 0
0 0 . . 1 23 35
0 0 --- 12 30
0 0 - 6.5 10.5 24
o 0 13 15 40
0.1 0.1 12 20 38
o 0 2.5 1.8 54
0 0 1.1 1.8 20
0 0 . 12 15 20
0.1 0.5 -- 70 45 81
0 53 48 82 60
0 9.5 --- 38.5 27.5 48.5
0 0.3 - 8.0 16.3 27.5
0 1.0--- 16 10 27
2 13 18 25
0 --- 0.1 0.1 4
0 -- 0.1 0 0
0 47.3 68 222
o 0-- 0 5 10
0.1 0.1 - 17 7.5 12
0 0.1 15 14 41
0 2.5 33 2.8 101
0 8 - 50 28 52
0 1.8--- 40 7 70
0 1.1 15 3.8 47
0 1 --- 30 7 31
0 5 29 23 53
0 0.2 0.2 6
3 3 16 28
8. 29 6 65
6 46 22 32
O 15 25 10 16
0 0 -- 5 10 15
Roberia bacteria natural inhibitor Robena bacteria
Lli .li!
F-
tJ
8 100 192 10
o 33 64 38
0 38 148 35
44 148 63 67 205 15 9.5 153 280 285 230 78
0 0 3 8 48 40 0 0 0 14 29 27
0.5 16 10 15 81 24 0 0 6.5 12 68
0 0 7.8 3.3 20.3 46.5 0 0 0 0 9.5 12.5
5/6
62
0
0
--
0
0.1
0.5
0
5/14
70
4
0
--
0.5
6
5
0
5/20
76
0
0
---
0
0.5
2
0
5/26
82
0
0
.--
0
6
5
o
6/3
90
0
8
.--
4
12
11
6/12
99
0
0
2
9
11
Table 34 - Ferrous Iron Along Horizontal System (ppm)
-------
Date and
Days After
Adding
Inhibitor
11/12 0
11/14 2
11/21 9
11/27 15
12/3 21
12/10 28
12/18 36
1/2/69 51
1/9 58
1/16 65
1/23 72
1/30 79
2/6 87
2/13 94
2/20 101
2/27 108
3/5 114
3/14 123
3/20 129
3/27 136
4/3 143
4/11 151
4/17 157
4/25 165
5/1 171
5/8 178
5/15 185
5/22 192
5/29 199
6/6 207
6/12 213
6/19 220
6/26 227
Robena bacteria natural inhibitor
1-1 1-2 13 1-4 1-5 16 17 18 1-9
3 3 4
4 4 4
4 5 6
4-5 4-5 45
3 3 3 3 3 4
3 3 3 3 3 3
45 45 45 45 2 2
3-4 3-4 3-4 5 3-4 3-4
3-4 3-4 3-4 3-4 3-4 3-4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
6 6 11 6 5 4
4 4 5 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
3 3 3 3 3 3
3 3 3 3 3 3
3-4 3-4 3-4 34 5 5
3 3 3 3 5 5
3 4 3 4 5 5
3-4 3-4 3-4 3-4 3-4 4
3 3 3 4 4 4
3 3 3 3 5 5
3-4 34 34 34 4 4
3-4 34 34 3-4 34 5
34 34 34 3-4 5 5
3-4 3-4 3-4 34 3-4 3-4
3 3 3 3 3 3
3 3 3 3 5 5
5 3 3 3 3 3
3 3 3 3 3 3
21 22 23 24 2-5 26 27 2-8 2-9
3 3 4
4 4 4
6 4 5
45 45 45
3 3 3 3 3
3 3 3 3 3 3
45 4-5 45 4-5 4-5 4-5
3-4 3-4
6 3-4 5 3-4 3-4 3-4
6 4 5 5 4 4
6 6 6 6 6 6
6 4 5 4 4 4
4 6 6 4 4 4
8 5 6 6 6 4
5 4 5 7 4 5
4 4 5 4 4 4
4 4 4 4 4 4
3 3 3 3 3 3
3 3 3 3 3 -- 3
34 3-4 5 3-4 3-4 4
5 3 4 4 3 3
5 4 5 5 4 4
4 3-4 5 3-4 3-4 3-4
4 4 4 4 3 4
3 3 4 3 3 3
4 3-4 3-4 4 4 5
34 3-4 3-4 34 3-4 3-4
34 34 5 5 3-4 5
3-4 3-4 34 3-4 3-4 3-4
3 3 3 3 3 3
5 3 4 3 3 5
5 3 3 3 5 5
3 3 3 3 3 3
Table 35 - Oxidation Times of Bacteria From Along
Horizontal System (days)
H
Robena bacteria
-------
Robena bacteria natural inhibitor
3.0 3.2 3.1 3.1 2.9 3.0 3.0 3.6 6.7
3.0 3.1 3.1 3.I 3.0 3.0 3.1 3.4 4.2
3.0 3.0 2.8 3.1 3.0 3.0 3.0 3.1 3.8
4.8 3.4 3.3 3.2 3.2 3.0 3.3 3.2 3.4
3.8 3.6 3.4 3.2 2.9 3.0 3.2 3.2 3.5
3.0 2.7 2.8 2.6 2.6 2.9 2.9 2.9 3.1
2.9 3.3 3.2 3.2 3.0 3.9 3.3 2.9 3.1
3.4 3.4 3.0 2,8 2.7 2.8 3.0 2.8 3.0
3.0 3.0 2.9 2.1 2.6 2.8 2.9 2.9 2.8
1/2 51 3.2 3.2 3.1 3.0 2.9 3.1 3.3 3.0 3.1
1/13 62 3.1 2.9 2.8 2.7 2.6 2.7 2.9 2.1 2.1
1/20 69 3.2 3.0 2.9 2.7 2.6 2.6 2.7 2.7 2.6
1/27 76 3.2 3.0 2.9 2.8 2.6 2.6 2.7 2.6 2.6
2/5 85
2/11 91
2/12 92
2/20 100
2/26 106
3/5
3/12
3/18
3/25
4/1
4/8
4/15
4/22
4/29
5/6 62 3.4 2.9 - 2.9 2.6 2.5
5/14 10 3.12 3.0 2.95 2.65 2.6
5/20 76 3.45 3.0 - 2.95 2.6 2.6
5/26 82 3.65 3.12 3.0 2.7 2.6
Robena bacteria
11 2-2 2-3 2-4 2-5 2-U 2-F -Y
3.0 3.6 2.0 2.0 2.6 3.0 3.0 3.3 5.4
6.2 3.4 2.4 2.8 2.7 2.7 3.0 3.) 3.3
6.5 3.1 3.0 3.2 2.7 2.7 2.9 3.0 3.0
2.2 2.6 3.0 3.2 2.8 2.8 2.9 3.0 3.0
3.2 2.8 3.2 3.4 3.0 3.0 3.1 3.1 3.1
3.7 2.7 3.0 3.0 2.7 2.8 2.8 2.8 2.8
6.3 3.4 3.6 3.6 3.2 3.1 3.2 2.8 2.8
6.3 3.8 3.5 3.2 2.9 2.9 3.0 2.8 2.9
6.3 3.5 3.3 3.2 3.0 3.0 3.0 2.8 2.9
6.6 3.6 3.6 3.5 3.2 3.2 3.1 3.0 3.0
6.2 3.5 3.3 3.2 3.0 2.9 2.9 2.9 2.9
6.1 3.? 3.6 3.6 3.1 3.1 3.0 2.8 2.8
4.5 3.7 3.6 3.6 3.2 3.1 3.0 2.9 2.9
3.7 2.7 2.9 2.6 2.4
3.95 2.8 3.15 2.75 2.35
4.95 2.6 2.75 2.6 2.25
5.55 2.7 2.65 2.6 2.4
Oat. and
Day. After
Adding
Inhibiter
11/11 1
11/15 3
11/19 7
11/25 13
11/26 14
12/4 22
12/9 27
12/12 30
12/16 34
Tap
!izQ.
7.1
7.
7.1
7.0
7.0
7.0
3.2 2.9 2.4 2.5 2.3 2.4 3.1 2.8 2.9 2.5 2.2 2.1
3.95 3.5 2.9 2.65 2.25 3.95 3.55 3.4 2.95 2.6
3.9 3.6 3.4 3.4 3.2 3.1 3.3 3.0 2.8 5.4 4.1 3.9 3.9 3.6 3.4 3.5 3.1 3.2
6.3 3.7 6.8 3.1 3.0 6.8 6.8 7.0 2.9 4.9 3.4 6.9 6.2 3.6 6.6 6.3 7.0 6.3
6.2 3.7 2.8 2.6 2.4 4.0 4.2 5.6 3.3 2.6
0 5.8 3.6 2.8 2.7 2.7 4.2 3.6 3.0 2.9 2.5
7 4.0 3.8 -- 2.8 2.8 2.7 3.5 3.5 3.2 3.2 2.8
13 4.8 3.65 - 3.4 3.1 - 2.95 4.45 3.72 3.35 3.2 2.87
20 3.5 2.9 265 245 2.3 3.5 2.65 2.5 2.42 2.15
27 3.25 2.95 - 2.8 2.6 - 2.45 3.7 2.9 2.6 2.5 2.3
34 3.35 2.95 2.95 2.75 2.6 3.72 3.1 2.8 2.65 2.4
41 3.4 3.0 3.0 2.62 2.55 4.35 2.75 2.9 2.6 2.3
48 3.25 2.9 2.7 2.5 2.4 3.7 2.7 2.6 2.55 2.3
55 3.2 2.72 - 2.45 2.3 2.22 3.5 2.7 2.7 2.5 2.2
6/3 90 3.9 3.1 2.9 2.6 2.45 5.45 2.95 2.8 2.6 2.45
6/12 99 6.8 6.9 6.7 6.55 2.55 6.7 5.9 6.75 6.65 2.5
Table 36 - pH Along Horizontal System
-------
Date and
Days After
Adding
Inhibitor
11/11 1
11/21 9
11/26 14
12/3 21
12/9 27
12/16 34
12/26 44
iZ 13 1-4 15
2225
825
850
675 1675 1950 3125 3675
275 100 350 425 650
300 300 650 950 1400
100 275 600 950 1575
2/5 85 100 100 --- 275 475
2/11 91 140 150 275 250
2/18 98 100 275 950 375
2/26 106 200 200 --- 400 575
I-b 1-7 18 1-9
1860
1425
1175
2150
1375
1675
2200
2450
5050
3625
4150
3250
2400
1125
1050
21 22 23 24 25
1400 2750
3250 1425
700 1500
1250 1775 1975 1750 1525
1250 150 150 275 475
100 150 300 400 600
110 85 75 200 400
27 28 29
1700
1125
1800
2800
1700
2175
2000
6/3 90 125 250
6/12 99 950 1000
50 295
850 375
500 120 100
500 1000 600
250 300
875 750
410
575
11
500
400
325
Robena bacteria natural inhibitor
Robena bacteria
a
0
1/8 57 550 800 1975 1825
1/14 63 125 110 - 115 2175
1/21 70 150 275 - 875 1450
1/28 77 100 450 950 1550
26
3/5 0 150 150
3/11 6 150 75
3/18 13 50 25
3/25 20 100 125
- - - 300 450
250 600
25 425
175 475
4/1
4/8
4/15
4/22
4/29
125 125 275 550 2525
135 135 - 200 550 2175
125 115 100 260 475
50 50 -- 250 275 825
125 150 140 200 550
125 275 150 225 425
250 950 750 750 850
150 125 -- 300 400 850
100 100 - 200 350 1000
125 50 - 140 215 650
1000 125 --- 160 375 850
125 250 - 425 475 1025
27 150 200 - 140 450
34 100 500 - 250 350
41 125 60 135 300
48 125 100 --- 300 550
55 50 175 --- 700 825
5/6 62 150 135
5/14 70 50 125
5/20 76 125 125
5/26 82 125 100
1000
925
750
750
600
500
275
575
1000
250
175
400
125
125 250
125 225
150 275
100 150
400
425
150
425
150
150 175
125 100
145 350
125 275
150 300
60 30
175 175
125 425
125 275
600
550
400
500
100
1075
1000
850
850
1125
600
625
850
710
90 175
50 40
265 300
125 125
Table 37 - Sulfates Along Horizontal System (ppm)
-------
Date and
Days After
Adding
Inhibitor
to 2.4 $ 5
F rrobaci 11 us
sulfooxidans natural inMbltor
I- .
0
11/19
12/4
12/9
12/18
1/2/69
1/9
1/16
1/23
1/31
2/6
2/14
2/20
2/27
3/6
3/13
3/20
-8
7
12
21
36
43
50
57
65
71
79
85
92
99
106
113
1-1 1-2 1-3 1-4 15 1-6 17
4 ,1 -- 86.3 65.0 -
0.5 --- 4.7 --- 5.7 --- 5.2
5,7 --- 6.2 --- 5.2 -- 3.6
2.6 -.- 7.3 --- 47.8 --- 17.7
19.0 - 28.6 - 17.7 - 8.3
--- 0.0 --- 0.2 -- 26.8 9.9
--- 1.0 2.1 25.0 56.0
0.0 0.3 125.0 48.0
0.0 0.2 20.3 108.0 63.0
7.8 1.8 13.0 -- 114.8 56.7
0.0 0.0 0.0 136.0 68.0
8.3 1.8 33.3 267.0
0.5 10.9 137.0 285.0 325.0
0.5 61.4 158.0 545.0 545.0
0.0 56.0 -- 3.6 -- 138.0 31.0
0.0 0.0 --- 6.8 58.0 -- 20.0
2-1 2-2
10.9 ---
4.7 --
2.6 ---
8.3 -
0.2 -
- 5.2
--- 0.2
0.3 0.0
0.0 0.0
2.3 3.1
0.0 0.0
--- 0.0
0.3 0.5
0.0 5.2
0.3 7.5
5.2 0.3
23 2-4
34.3 ---
1.2 ---
3.6 --
3.1 0.5
15.6 1.0
--- 0.0
-- 0.0
--- 4.0
0.0 2.3
5.2 6.5
2.6 3,6
4.7 6.0
4.7 7.8
15.6 7.8
67.0 30.0
44.0 24.0
2-5 2-6 2-7
24.4
13.0 -- 14.6
4.7 -- 3.4
23.1
11.2
13.8
27.0
26.0
42.6
44.5
52.0
83.0
101.0
230.0
119.0
117.0
11/19
12/4
12/9
12/18
1/2/69
1/9
1/16
1/25
1/31
2/6
2/14
2/20
2/27
316
3/13
-8
7
12
21
36
43
50
57
65
71
79
85
92
99
106
Ferrobaclllus ferrooxidans no Inhibitor
Ferrobacillus
ferrooxidans
natural Inhibitor
2:1 2: 1 2 :
121.0
0.0 --- 8.0
2.9
10.7
- 5.5
94.0
2.0
36.0
13.0
109.0
0.5 268.0 -- 215.0
230.0
1.8
L i!
i
15.0
12.2
17.0
1.0
-- 3.0
--
3.0 -- 10.0
6.2
8.3
---
51.5
---
3.0 --- 11.0
0.0
8.8
15.6
18.0
17.0
12.0
13.5
15.6
85.0
75.0
17.0
22.9
18.0
13.0
21.0
34.3
98.0
51.0
52.0
46.7
62.4
253.0
34.0
68.0
417.0
587.0
0.0
10.0
-
0.2
0.1
4.4
23.4
0.0
0.1
1.6
0.0
0.0
0.0
--
---
0.0
---
0.2
0.3
2.3
0.0
3.1
0.0
0.1
0.0
0.0
10.0
0.5
--
5.2
0.5
7.3
-
-
---
--
0.5
0.8
0.3
4.2
2.6
0.1
5.7
0.0
0.5 54.6
79.0 155.0
91.0 113.0
128.0 85.0
125.0
-- 73.0 80.0
78.5
67.0 107.0
106.0 210.0
--- 137.0 76.0
70.0 114.0
- 94.9 41.0
-- 36.0 43.0
Table 38 - Total Iron (ppm) Through Vertical Spray System
-------
No added bacteria
51 52 53 54 55 56 57
61 62 63 64
I- . ,
I-
6-5 6-6 6-7
3.0
---
23.1
111.0
--
9.0
--
--
428.0
600.0
58.0
107.0
320.0
355.0
166.0
151.0
---
---
280.0
275.0
43.0
535.0
343.0
450.0
465.0
136.0
31.0
9.0
11.4
22.4
255.0
75.0
61.4
113.0
281.0
265.0
260.0
550.0
9.0
114.0
51.0
80.0
70.2
130.0
257.0
245.0
335.0
535.0
151.0
60.0
13.0
13.0
7.3
343.0
55.0
80.0
91.0
11.0
32.0
69.0
133.0
116.0
275.0
20.0
33.0
1.8
0.0
0.0
10.4
0.3
0.3
2.3
0.0
0.3
0.1
0.0
0.0
0.0
0.2
0.2
0.0
1.8
2.3
0.0
0.0
0.1
0.0
0.5
0.0
43.0
1.0
1.0
4.4
0.0
0.3
0.0
1.0
0.2
0.0
3.1
0.1
1.0
10.4
8.3
0.2
20.0
0.5
1.0
8.8
3.6
11.0
13.0
17.7
26.0
.28.8
26.0
32.0
0.0
42.0
Date and
Days After
Adding
Inhibitor
Mixed species natural
inhibitor
Mixed
species no Inhibitor
to 2,4 & 5
11/19 -8
12/4 7
0.0
--
12/9 12
-
3.0
12/18 21
--
-
1/2/69 36
225.0
1/9 43
--
328.0
1/16 50
--
1/23 51
0.0
1/31 65
2/6 71
2/14 79
--
-
2/20 85
---
2/27 92
395.0
3/6 99
---
---
3/13 106
--
3/20 113
0.0
-
71
7-6
7-7
7-2
73.
74 75
11/19 8
86.0
-
54.0
-- -
61.0
12/4
12/9 12
3.0
3.0
---
87.0
20.0
116.0
- 49.0
--
12/18 21
1/2/69 36
3.4
0.3
--
-
49.7
2.6
-- 47.8
37.4
-
--
1/9
-
0.3
-
4.2 --
15.0
1/16 50
1/23 51
1/31 65
2/6 71
0.0
0.0
3.1
1.0
0.0
0.0
2.3
--
16.6 23.7
17.0
22.0 31.0
28.6 44.5
-
--
---
--
1.6
2.0
2.8
19.8
17.0
8.8
7.8
6.8
41.0
26.0
35.0
94.0
31.0
31.0
54.0
54.0
38.0
14.6
13.0
23.1
16.0
55.0
62.1
Table 38
- Total Iron (ppm) Through Vertical Spray System (Cont.)
-------
Ferrobacillus sulfooxidans no inhibitor
11 12 13 14 15 16 11
Date and
Days After
Adding
Inhibitor
to 2 4 & 5
11/19 -8
12/4 1
12/9 12
0.0
2.5
---
--
56.0
0.0
4.0
.- -
---
0.0
0.0
48.0
- 0.0
0.0
12/18 21
1/2/69 36
0.0
0.2
---
---
0.0
0.0
--
--
40.0
0.0
-- 0.0
1/9 43
0.0
0.0
---
0.0
---
2.5 1.0
1/16 SO
---
o.o
0.0.
0.6 17.8
1/23 57
0.0
0.0
6.0
-- 3.3
1/31 65
0.0
0.0
1.0
3.0 14.5
2/6 71
2/14 79
0.0
0.0
0.0
0.0
0.5
0.0
--
7.5
-- 12.5
11.0 22.0
2/21 86
0.1
0.3
0.5
113.0
2/27 92
3/6 99
0.0
0.0
0.0
7.0
2.0
8.5
13.0
8.0 58.0
92.5
3/13 106
0.0
5.5
-
0.0
--S
18.0 9.5
3/20 113
0.0
0.0
-
0.0
10.0
--- 6.0
Ia
1.,
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.8
0.0
4.5
0.0
1.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
13.0
8.5
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
0.0
7.5
3.5
0.0
0.0
0.0
2.0
3.5
1.8
13.0
9.5
15.0
28.0
2.0
74.0
7.5
38.0
Ferrobaclllus ferrooxldans no inhIbitor
3-1 3-2 3-3 3-4 3-5 36 37
Ferrobacillus u1fooxidans natural inhibitor
21 22 2-3 2-4 2-5 2-6 21
17.8
0.0
0.5
Ferrobacillus ferrooxidan natural inhibitor
4-1 4-2 4-3 4-4 4-5 4-6 4-7
7.5 35.0
0.0 --- 0.0 - 0.0 -- 2.5
0.0 -- 5.5 -- 0.0 2.0
0.0 --- 0.0 0.0
0.0 -- 0.0 3.3 22.5
0.0 0.2 12.5 27.5
0.0 --- 0.0 16.8 60.5
0.0 0.0 --- 0.0 26.5
0.3 0.3 -- 0.3 -- 27.0 34.0
0.0 0.0 0.0 0.0 25 .5
0.0 0.0 0.0 14.0 47.5
0.0 1.0 14.5 77.0
0.0 0.0 --- 0.0 --- 5.5 13.0
0.0 0.0 -- 0.0 --- 5.0 33.5
0.0 0.0 --- 0.5 --- 15.5 15.5
0.0 0.0 -- 0.0 --- 8.5 13.0
11/19
12/4
12/9
12/18
1/2/69
1/9
1/16
1/23
1/31
2/6
2/14
2/20
2/26
3/6
3/13
3/20
-8
7
12
21
36
43
50
57
65
71
19
85
92
99
106
113
63.0
0.0 --- 0.0
0.0
0.0
0.0
3.3 -
0.0
0.0 2.3
0.0 5.0
10.5
.0.0
0.0
78.0
10.0
8.0
0.0
0.0
0.3
0.2
0.5
3.5 0.3
0.1
0.1
16.5
6.1 2.3
0.1
8.0
3.5
0.1
16.5
61.0
6.0
5.3
0.3
7.5
33.8
4.5
14.3
10.5
28.0
72.5
1.8
20.0
160.0
108.0
Table 39 - Ferrous Iron (ppm) Through Vertical Spray System
-------
Date and
Days After
Adding
Inhibitor
to 2,4 & 5
11/19 -8
12/4 7
12/9 12
12/18 21
1/2/69 36
1/9 43
1/16 50
1/23 57
1/31 65
2/6 71
2/14 79
2/20 85
2/27 92
3/6 99
3/13 106
3/20 113
51 52 53
5.0
0.0
0.0
9.8
17.5
3.5
8.3
6.0
25.0
0.0
54 55
8.0
1.75
1.0
0.0
1.3
14.0
19.8 14.8
17.0 10.0
11.5 7.0
6.0
9.5 7.5
7.0 7.5
7.5 5.5
28.0 28.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
65 6-6 67
1.8
0.0 - 0.0
0.0 --- 0.0
0.0
0.0 -- 0.5
0.0 0.3
5.0
1.0
3.0 -- 2.0
2.5
4.5 --- 14.5
3.0 -- 5.8
2.0 -- 1.8
3.5 2.5
14.0
5.0 14.0
Mixed soecles natural Inhibitor
I-J
(A,
Mixed species no lnhibttor
61 6-2 63 64
7.5
0.0
0.0
0.0
4.5
7.0
9.0
56 57
26.0
5.5
4.5 5.0
0.0 0.5
4.0 17.0
5.5 13.3
36.5
6.0 0.3
8.0 6.8
11.0 19.0
8.0 43.5
8.0 29.5
7.5 148.0
32.0 8.5
22.0 19.0 -- 38.0 11.0
0.2
0.2
0.0
1.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.2
2.0
0.0
0.0
0.0
0.0
0.0
4.0
0.0
No added bacteria
7-1 72 7L3 74 75 76 77
11/19 -8
4.5
-
1.8
-
-
3.0
12/4 7
0.0
-
3.0
-
3.0
--
4.0
12/9 12
0.0
---
5.0
---
6.0
-
6.0
12/18 21
0.0
-
0.5
1.0
1/2/69 36
0.0
---
0.0
-
0.5
--
0.6
1/9 43
--
0.3
---
0.3
-
0.8
1.8
1116 50
-
0.6
-
2.5
4.3
5.0
1/23 57
0.0
0.0
---
0.5
0.3
--
0.0
1/31 65
0.0
0.0
-
2.5
2.0
19.5
2/6 71
0.0
0.0
-
4.8
6.5
21.5
Table 39 - Ferrous Iron (ppm) Through Vertical Spray System (Cont.)
-------
Ferrobacillus sulfooxidans no inhibitor
11 12 13 14 15 16 17
Ferrobacillus sulfooxidans natural inhibitor
1 ._a
100
---
175
-
375
225
-
525
450
---
575
215
---
350
--
250
--
250
125
--
400 -
400
--
175
100
--
250
--
700
--
275
125
--
150 125
300
125
--
250
--
275
--
175
175
-
375 200
275
125
---
400
--
-
275
200
125
190
225
150
115
260
-
125
--
125
300
--
125
115
300
75
75
-
125
325
50
75
700
500
150
125
225
140
275
125
140
290
75
---
275
55
75
150
155
300
50
75
125
675
450
25
50
75
150
400
50
50
1150
--
135
150
225
550
125
150
425
1125
1050
150
150
150
150
450
125
200
775
1915
125
125
.200 125
850
50
300
400
625
575
325
600
275
500 300
640
---
---
375
125
160
450 275
750
Date and
Days After
Adding
Inhibitor
to 2 4 & 5
11/19 8
12/4 7
12/9 12
12/18 21
1/2/69 36
1/9 43
1/16 50
1/23 57
1/31 65
2/6 71
2/14 79
2/21 86
2/27 92
3/6 99
3/13 106
3/20 113
11/19 -8
12/4
12/9 12
12/18 21
1/2/69 36
1/9 43
1/16 50
1/23 57
1/31 65
2/6 71
2/14 79
2/20 85
2/27 92
3/6 99
3/13 106
3/20 113
Ferrobaclllus ferrooxidans natural inhibitor
Ferrobacillus ferrooxidans no Inhibitor
3-1 3-2 3-3 3-4 3-5 36 37
850 --- 1675 - 1075
175 --- 275 --- 700 -- 375
125 325 325
140 125 --- 150
260 275 -- 285
150 --- 350
450 225 175
250 - 300
225 290 275 325
200 250 300
0.1 -- 28
425 550 900
140 525 -- 200 425 -- 275
150 --- 400
275 300 --- 1250
150 270 - 1975
4-6 47
4-1 4-2 4-3 4-4
100 -- 1450
275 -- 275
100 --- 275
140 --- 160
200 -- 260 - -
135 650
175 225
150 75 75
75 125 --- 500
75 290 150 275
14
135 275
125 150 - 300
40 125 --- 275
300 290 -- 425
125 155 - 225
45
825
300
130
140
825
400
600
475
525
550
300
675
375
375
125
450
1425
650
700
400
500
400
47.5
725
400
450
270
300
Table 40 - Sulfates (ppm) Through Vertical Spray System
-------
Date and
Days After
Adding
Inhibitor
to 2.4 & 5
11/19 -8
12/4 7
12/9 12
12/18 21
1/2/69 36
1/9 43
1/16 50
1/23 57
1/31 65
2/6 71
2/14 79
2/20 85
2/27 92
3/6 99
3/13 106
3/20 113
6-1
225
175
150
125
110
75
175
150
150
100
75
125
125
Mixed species no inhibitor
6-4 65 66
1150
450
200
140
260
125 175
250 300
175 350
290 350
4.5
275
150 275
200 225
250
125 -- 500
6-2 :I
1075
275
265
125
100
160
125 125
150
175
125
220 150
140
65
275
150
67
425
125
150
150
250
250
250
290
14.5
300
475
650
300
375
No added bacteria
72 73 74 75 7-6 77
425 --- 1000
800 --- 800 --- 650
100 - 525 --- 775
240 -- 500 -- 375
250 - 525 - 400
135 200 200 350
250 150 275 300
75 150 325 275
175 350 425 475
115 275 400 425
I a
ljJ
U,
f4ixed soecies natural inhibitor
1200 -- 1275 1275
125 250 - 400 125
265 -- 600 - 550 275
350 -- 425 - 150
1725 1500 1225 875
1210 725 700
1300 1250 --- 675 1400
1175 1025 650
400 600 425 550 270
500 550 350 350 275
6 11 19
1700 1000 950 725
750 -- 1125 950 750 425
1000 1250 700 1275 1400
950 1300 1675 1550 300
225 --- 400 500 --- 720 260
71
11/19
-8
600
12/4
7
300
12/9
12
12/18
21
150
125
1/2/69
36
1/9
43
---
1/16
50
--
1/23
57
1/31
65
225
Table 40 - Sulfates (ppm) Through Vertical Spray System (Cont..)
-------
-a
Date and
Days After
Adding
Inhibitor
2 4 & 5
Ferrobecillus sulfooxidans no inhibitor
Ferrobacillus
sulfooxidans
natural Inhibitor
11 12 13 4 5
16 1.7
21 2-2
23 24
2-5
2-6 .Z.
11/25 -2
5.9 3.3 3.0 3.1 3.4
3.8 4.0
3.2 3.1
3.3 3.5
3.8
3.8 3.7
12/4 7
6.6 3.3 3.8 3.8 4.0
4.1 4.1
4.2 3.2
3.1 3.3
3.5
5.8 3.9
12/12 15
7.0 3.1 4.3 4.2 3.9
3.9 5.7
6.4 3.1
4.1 4.5
--
6.0 5.5
12/20 23
1/2/69 36
1/13 47
1/20 54
1/23 57
1/27 61
2/6 71
6.0 3.0 3.2 3.3 3.2
6.3 4.7 4.0 4.4 3.6
6.5 6.3 3.5 4.2 3.2
5.8 5.7 3.3 -- 3.1
6.0 5.2 3.2 3.3 3.0
2.9
3.5 3.5
-- 4.0
3.4 3.6
3.2 3.2
2.95
3.0 3.1
6.5 3.2
6.9 3.3
6.8 3.8
6.0 3.7
6.4 4.3
3.5 3.7
3.8 4.8
3.9 4.6
4.2 4.1
3.8 3.6
3.7
3.5
3.4
2.9
3.3
2.85
2/13 78
2/27 92
3/6 99
3/20 113
5.3 3.5 3.2 --- 2.8
3.2 3.1
2.45
2.7
2.75
6.1 4.3
*
3.6 3.6
3.3
2.6
2.95
2.1
Ferrobacillus ferrooxidans no
inhibitor
Ferrobacillus
ferrooxidans
natural
inhibitor
Infl uent
7.1
7.2
7.1
7.1
7.0
7.1
7.1
7.2
7.1
7.1
7.0
7.1
1L
11/25
11/4
12/12
12/20
1/2/69
1/13
1/20
1/23
1/27
2/6
2/13
2/27
3/20
-2
7
15
23
36
47
54
57
61
71
78
92
113
--
6.5
---
--
---
3.0
4.7
3.3
2.9
3.1
3.3
3.8
--
--
-
3.4
3.1
3.1
3.5
3.5
3.5
3.3
3.4
3.0
3.2
3.3
3.2
3.9
3.9
3.7
3.4
3.3
3.1
3.4
5.9
6.0
---
6.6
6.6
4.7
4.1
3.6
3.4
3.7
5.8
5.5
3.6
3.6
3.3
3.2
2.75
3.1
2.8
3.2
2.75
3.0
2.5
4.9
4.2
3.2
-
6.8
6.6
6.4
4.5
3.1
3.8
3.2
4.0
4.2
4.1
-
4.1
3.9
3.1
3.2
3.3
3.8
4.0
3.6
3.6
3.4
3.5
3.5
4.0
6.3
5.9
5.9
3.7
3.7
3.5
4.0
4.0
---
---
--
3.3
3.4
3.6
4.3
3.4
3.3
3.2
3.1
3.0
3.2
3.4
4.4
5.6
3.3
3.3
3.1
3.2
2.6
3.0
2.7
3.2
2.7
2.95
2.55
Table 41 - pH Through Vertical Spray System
-------
Oate and
Days Mixed sped.. natursi inhibitor Mixed specIes no inhibitor Influent
Inhtbl tar
11/25 2 - 2.5 3.0 3.0 4.5 3.4 4.5 6.7 3.0 3.5 3.4 3.5 4.8 5.0
12/4 7 5.5 2.9 3.2 3.0 5.6 3.5 5.1 6.6 3.2 6.1 3.2 3.3 3.4 5.2
12/12 15 4.1 3.7 3.9 4.4 6.6 3.5 3.9 5.2 5.1 5.8 6.0 7.1
12/20 23 - 3.4 3.6 3.3 4.4 3.6 7.1 4.3 6.5 5.8 3.9 6.0 6.1 7.2
1/2/69 36 3.0 3.2 3.3 3.4 3.5 7.1 5.9 7.0 6.6 3.8 4.6 4.2 7.1
1/13 47 2.9 2.9 3.1 3.1 3.3 6.6 6.8 6.7 6.8 3.6 5.5 3.8 7.1
1120 54 2.7 2.7 3.0 3.0 3.2 6.7 6.5 6.4 5.6 3.5 4.4 3.6 7.0
1/23 2.85 3.0
1/27 61 2.9 2.7 2.7 2.8 2.9 3.0 6.8 5.8 6.0 4.2 3.3 4.0 3.5 7.1
2/6 71 2.9 3.0
2/13 78 - 3.0 3.1 2.9 3.1 3.1 3.2 6.6 4.5 4.3 3.9 3.4 3.6 3.4
2/27 92 2.55 2.8
3/6 99 2.8 3.0
3/20 113 2.7 25
Mo added bacteria
7-1 7-2 7-3 7-4 75 7-6 77
11/25 2 3.7 3.4 3.4 3.0 3.0 3.1 3.1
12/4 7 3.5 3.1 3.1 2.9 2 .9 3.0 3.1
12/12 15 5.7 4.7 5.4 3.2 3.2 3.2 3.5
12/20 23 6.6 5.0 3.6 3.3 3.4 3.2 3.5
1/2/69 36 4.3 4.5 4.7 3.5 3.5 3.2 3.7
1/13 47 6.3 6.7 4.4 3.5 3.3 3.5 3.5
1/20 54 6.0 6.6 3.7 3.3 3.3 3.3 3.2
1/23 57 2.85
1/27 61 6.0 4.0 3.7 4.1 3.2 3.2 3.3
Table 41 p1-I Through Vertical Spray System (Cont.)
-------
TABLE 42 - TOTAL ACIDITY THROUGH VERTICAL SPRAY SYSTEM (ppm)
Date and
Days After
Adding
Inhibitor Tank Tank Tank Tank Tank Tank Tank
To 2, 4 & 5 No.1 No.2 No.3 No.4 No.5 No.6 No.7
11/19 8 140 10 10 170 165 50 90
12/4 7 --- 55 180 30 30 -2 140
_ 12/9 12 20 20 15 _5 10 0 190
12/18 21 110 155 105 235 60 20 225
1/2 36 25 35 140 275 295 10 35
1/23 57 260 195 280 625 400 120 205
1/31 65 300 330 315 475 125 105 320
2/6 71 250 235 280 460 230 120 320
2/14 79 440 355 380 560 405 270
2/20 85 60 105 990 660 690 292
2/27 92 1475 485 255 335 230 580
3/6 99 2230 650 430 500 1750 510
3/13 106 215 485 1505 280 150 215
3/20 113 175 550 1075 335 145 290
-------
BI5LIOGRAPIIIC: iSA Renearci: Corporation. tffoct of Viable ACCESSION NO;
Aitihactorial Agents on line Drainage::.
KEY WORDS:
ABSTRACT? A program was carried out to ol:aractorize the nature Acid Mine Water
of the active agents in curtai n natural waters that had previously
bee:: fpund to i nI:xl:lt batt:rtal production of acid in straaos iron Dacturda-
throug h suboerged puns of coal iu plastic containers in the
laboratory. A farther minor program was nndcrtalc:: to test Wine Acids
tI:u feasibility of application of tine inl:ibitory principle to
a:: actual coal nix::: situation where acid was bui::y produced. Mine Drainage
In tine ni::n study, two inn: ulations of tl:u natural wafer prcwi Acid Streams
ousiy found to bu ininibitox y were made in tine amou::t of about
14,000 gallons each to n wn riced-out region of Robe::a mine in Aerobic Bacteria
Dreene County, Pennsylvani: . After tine first inoculation was
made: laboratory tests shosed absence of inhibitory power in Mine Wastes
ti:e water at tine tine it wx s collected: and tine water used for
tine second test was sinilanly found to be only weakly inhibttory. coal Mine Wastes
Rn effect was noted in the drai::age of thu test sit:: over periods
of several months after sack: i:noculattnn. In addition to the Acid Bacteria
possibility of tine weakness or absence of tine inhibitory prin-
ciple, it is new beliuved that poor topngraplny of tine floor of Chemical Wastes
the nine of thu test situ sight render application: of inl:ii:itor
inneffoctive. A sore promising test site is believed to he at Pyrtte
Karen isin:e, alan ix: seuthwastnrn Pennnsylvania.
Water Pollution
In effects to cI:araeterize tl:e nature tinu inhibitor, earlier, Sources
Coal Mines
BIBLIOCRAPHIC: NSA Research Corporation, Effect of Viable ACCESSION 110:
lis:tibaeterial Agents on 1-Itne Drainages.
KEY WORDS:
ABSTRACT: A program was carried out to characterize the nature Acid Mi:ne Water
of the active agents I:: eertsta natural waters that had previously
been found to inhibit hacte±ial production of acid in streams Iron Bacteria
througi: submerged piles of coal in plastic containers is the
laberatery. - A further nni::er program was u::dertatnn: to test Mine Acids
the feasibility of application of the inhibitory principle to
as actual coal mine situation wi:ere aetd was being produced. Mine Drainage
In the mine study, two inoculations of the natural water pravi Acid Streams
ously found to he inhibitory were made in the amount of about
14,000 gallons each to a worked--out region of Robena mine in Aerobic Bacteria
Greeno County, Pennsylvania. After the first inoculation was
madu, laboratory tests showed absence of inhibitory power in Mine Wastes
the water at the time it wan collected: and tl:o water used for
thu second test was similarly found to be only weakly inhibitory. Coal Mine Wastes
No effect was noted in - the drainage of tine test site over periods
of sevural months after each inoculation. In addition to tine Acid Bacteria
possibility of tl:o weakness or absence of tl:e inl:ibitory priu -
ciple, it is mow believed that poor topography of tine fleer of Chemical Wastes
the mine of ti:e test site might render applicatics: of inhibitor -
ineffective. A more promising test site is believed to be at Pyrtte
Karen mine, also in southwestern Pennsylvania. -
- Water Pollution
In efforts to characterize the ::ature the i:nhibitor, earlier, Sourdes
Coal Mines
BIBLIOGRAPHIC: NSA Research Corporation, Effect of Viable ACCESSION NO:
Antibacterial Agents on Mime Drainages.
KEY WORDS:
ASSTRACT: A program was carried out to ei:aracterize the nature
of the active agents in certain natural waters that l:ad previously Acid Mime Water
been-found to inhibit baeterisl production of acid do streams
throsgin submerged pilus of coal in plastic containers is thu Iron Bacterie
laboralnryl A furtiner mimer program was undortatsn to test
tl:c feasibility of application of the inhibitory principle to Mime Acids
am actual coal mtno situation where acid was being produced.
- Mine Drainage
Is the mine study, two inoculations of the natural water previ-
ously found to be imhibitony were made in the amount of about Acid Streams
14,000 gallons cad: to a wcrtodout ,rc ien of Rnbena mime in
Greene County, Poi:ssylvanic. After the first inoculation was Aerobic Bacteria
made, laboratory tests shosed abso:nce of inhibitory power in
the water at ti:u time it ens collected; and the water used for Mine Wastes
tinu seconnd test- wa3 similan ly found to-bo only weakly imlititory. -
No effect was mound i:: tao drainage nf thu test site over poriods Coal I-line Wastes
of several ns)eths after earl: inoculation. In ddnlitiun to the
possibility of tine weahinean or abson:ce of the inhibitory prim-- Acid Bacteria
ciple, it is now believed I hat door topography of tine floor of
tine sin:e of the test site pinjint render application of in:hibitnr Cinemical Wastes
ineffective. A mnru prnmin ing test site is hulioved to he at -
1mm:: min:o, alno in snutlnwnstern Ponnssylvan:l 5 Pyr ito
I:: efforts to ohareeterize tine mature of thus inl:ibitnr, earlier, Mater Pollution
Sources
Coal l4ises
-------
u;tncpiicatud, laL.nratory c4erinwnts cor n du 1 ,licnlcsl on atrearsn Indeetrial Wuntes
tLrougi. tuiinergen acid-prcihsctit iilen of coal in plantin con
tainern. fnecujetioa, of both r aw and tr eated cuters previOusly wa, ,tes
found innihitory resultod i i. a docreanc in 1 ,rocioction of acid,
liuno iwni ire,, nod suifntoa and tn an ircrean,, iii di, Water Pollution
Aija Lament
Ptrains of Cauini,acter which arc bacteria characterized iy stalked
nppendagca were found in the natural inhibitory waters; and e l len Pollutants
adapted to acidic nnvironmthito nnd concoeti-ated ii; culture media,
they vero inoculutcil into laboratory coal j utes. they then induced Pollution Abete
inhibition of the acid production. Other adaptod ntratna of caulo i nert
banters obtained cemnercially alne tnduced ind,ibttton. lure
evidence was shown that Caulobacter inhibitorn night move down Ferrobacillu s
stream ii . a floodu;d lioricontat ntroan through pilea of coal refune
producing acid Thiobaciltus
Ferrooxidana
A group of nnnsitivity diske containing antibieticu made by various
species of Streptonyocs cn n scrcencd for uffectivoness against the Microbiology
iron-sulfur bacteria producing acid in circa. TI;o four epncioa of
Strc 1 itnnyces producing the antibiotice found to ho effective were nactericiden
aduutod to the acidic conditiose found in nins seniors and wore found
effocii-we against the acid-producing bacteria in test tube cultures Acidic Water
and on colid ecdie.
Mine Water
Water Pollution
Strip Mine Wastes
Mining
unreplicatrud. laboratory oIpnrlrlents wore duplicated on streams Industrial Wastes
through submerged acidproducing piles of coal in plastic con-
tainers. Inoculation of beth raw sod treated waters previously Wastos
found inhibitory rusutted In a decrease in production of acid,
dissolved iron and sulfates and in an increase in p 1 1. Water Pollution
Abe tenant
Strains of Cauiobaoter which are bacteria characterized by stalked
appendages were found in tie natural inhibitory watersi and uhnon Pollutants
adapted to acidic anvironnsinta and concentrated in culture media,
they were inoculated into laboratory cost pitea. f hey then induced Potteticn Abatn-
inhibition of the acid rroiuction. Other adapted strains of Caulo flent
bactors obtainad coeuncrcia)ly also induced inhibition. Sose
ovidnnce was shown that Caelobacter Inhibitora night rove down Perrobacittua
stream in a flooded I;ortao,tsl stress through piles of coal refuse
prodncing acid. thiobarilinis
rerrcoxidase
A group of sensitivity dia)s containing antibiotics made by various
species of Streptonyeoa wal; screeosd for effnc;ivociaas against the Microbiology
iron-auifur bacteria produs:i;;g acid in mdocs. The four atuecioa of
Streptosycnn producing the antibiotics found to he effective were lactericides
adapted to thu acidic conditions found in mine waters and were found
effective against the acid- producing bacteria in teat tube cultnrss Acidic Water
and on solid sadia.
Wine Water
Water Pollution
ttnip nine Paste
Mi;u ing
cnreplicated, laboratcry experiments ware duplicated on streams Industrial Waste
thrcnagl; subnergeui acid-producing piles of coal in plastic con-
tainers. Inoculation of both raw and treated waters previously saamtea
found inhibitory resulted In a decrounc In production of acid,
dissolved iron and sulfates and in an inereaue in p1 1. Water Pollution
Abatement
Strasns of Caulebacter which are bacteria characteriaed by stalked
appendages were foun in the natural inhibitory watern ; and when Pollutants
adapted to acidic enflromsests and csncentratod in culture media,
they were inoculated into laboratory coat puce, they then induced Pollution Abate-
inhibition of acid production. Other niapted ntrainn of Caulo nent
buotaur:; ohutainec eor.snnrcially also induced inhibition. Isse
suvidesce was shown that Caulohecter inhibitors might hove down- yerrobaciilua
ntrean in a flooded horizontal ntrean tL;rougl. puns of coal refuse
producing acid. thiobaciltua
Ferrooxidana
A group of sensitivity disks containing antibiotics made ly various
species of Stroptoeycca Wan screened for snifoctivonean agaiost the Microbiology
ironsulfur bacteria producing acid in mines. the four species of
Otreptoryces producing the antibiotics foucal to he nffectivo were Inactericidea
adapted to the acidic conditions found in mine waters and wure found
effective agai nst the acidproducing i,aot,.-ria in teat tube cultures Acidic Water
and en solid media,
line Water
Water Pollution
Strip Mine Wasto
Pining
------- |