...-_ StCtM
tnvirtwuYMnul Protection
Agency
Environmental Research
Laboratory
Dulutfi MN 55804
EPA-600 3 80-046
May 1980
and Development
Environmental
Effects of Western
Coal Surface Mining
Part VII.
Microbial Effect on the
Quality of Leach
Water from Eastern
Montana Coal Mine
Spoils
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-80-046
May 1980
ENVIRONMENTAL EFFECTS OF WESTERN COAL SURFACE MINING
PART VII - MICROBIAL EFFECT ON THE QUALITY OF LEACH WATER
FROM EASTERN MONTANA COAL MINE SPOILS
by
Patrick F. Kimble and Kenneth L. Temple
Department of Microbiology
Montana State University
Bozeman, Montana 59717
Grant No. R803950
Project Officer
Donald I. Mount
Environmental Research Laboratory - Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency. and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommendation
for use.
if
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FOREWORD
Some of the most severe effects of surface coal mining in eastern U.S.
have been a result of leaching of mine spoils especially where oxygen is
also available. If mine spoils can be properly placed to avoid such effects,
the impact of mining can be reduced.
This study is one of a series to assess the environmental impact of
western surfa~~ coal mining. In this report, the question of acid formation
potential from mine spoils was investigated. The toxicity of leachates to
algae was also assessed. While not complete enough for final answers, this
study does provide useful insight into mine spoil impacts in western regions
of the U.S.
J. David Yount, Ph.D.
Deputy Director
Environmental Research Laboratory-Duluth
iii
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ABSTRACT
. Eight test hole cores of overburden grab samples from the Bear Creek Study
Slte of the West Moorhead coal deposit in eastern Montana were received from
the U.S. Geological Survey. The samples were visually inspected for evidence
of mineralization. This was aided by an accompanying geological log for each
core. Each sample was ground prior to its analysis for lead content, pH value,
conductivity, and chemoautotrophic bacteria presence. The mineralization of
the core samples was quantitatively different for the strata, with no consis-
tent relation between physical and chemical descriptions.
Chemoautotrophic bacteria, both sulfur and iron oxidizing, were isolated
from a number of core samples. Difficulty was encountered in obtaining pure
cultures. Growth was not enhanced when organic compounds useful for some
Thiobacillus species were included in the medium. The fastidiousness of these
cultures does not typify sulfur or iron oxidizers in general, and therefore
is characteristic of these isolates. By contrast, a culture of iron oxidizing
bacteria isolated from a revegetation study site on the Colstrip coal deposit
in eastern Montana was similar to the typical iron oxidizing bacterium Thio-
bacillus ferrooxidans.
Leaching studies were performed on samples which exhibited a wide range of
lead concentrations, pH values, and conductivities. Samples were ground to <80
mesh. The study included the comparision of static and shaking conditions,
with and without added glucose, with and without an inoculum of sulfur and
iron oxidizing bacteria. Lead values in the leachates were roughly similar
and not proportional to the lead content of the core samples. Consequently,
autotrophic oxidation did not proceed at the rapid rate which occurs with
unbuffered high pyrite cores. Most strata did not develop low pH values on
leaching but a few strata did. Values as low as pH 1.61 were observed.
The algal bioassay procedure (U.S. EPA 1971) was used to determine the
possible toxic effect of the leachates using Selenastrum capricornutum
Printz, and a 1:10 dilution of the leachate. Growth of the algae was monitored
by fluorescence spectrophotometry on a daily basis. Some leachates produced
inhibition of algal growth explained by low pH values. The toxicity of
other leachates could not be explained but was not due to either pH or to lead.
For the majority of leachates no toxicity was observed, and some leachates
were stimulatory.
iv
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CONTENTS
F 0 rewa rd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. i i
Abs t ra ct. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i
F; 9 u re s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. v
Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. vi
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . viii
VII
VI II
I
Introduction.
. . . . . .
. . . . .
.........
. . . . .
I I
Conclusions. . . .
. . . .
.........
. . . .
. . . . .
I I I
Recommendations.
. . . . . . .
. . . . .
...........
IV Description of Study Area. .
. . . .
. . . .
.........
V Materials and Methods. . . . . . . . . . . .
. . . . . . . . .
VI
Med; a . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Glassware. . . . . . . . . . . . . . . . . . .
Samp 1 i ng . . . . . . . . . . . . . . . . . . . . . . . . . . .
Samp 1 e An a 1 ys is. . . . . . . . . . . . . . . . . . . . . . .
Leaching Studies. . . . . . . . . . . . . . . . . . . . . . .
Algal Bioassays . . . . . . . . . . . . . . . . . . . . . . .
Re s u 1 t s . . . . . . . . . . . . . . . . . . . . . . .
. . . . .
Samp 1 e An a 1 ys is. . . . . . . . . . . . . . . . . . . . . . .
Enrichment Cultures. . . . . . . . . . . . . . . . . . . . .
Algal Growth. . . . . . . . . . . . . . . . . . . . . . . . .
Leaching Studies. . . . . . . . . . . . . . . . . . . . . . .
Algal Bioassays of Leachates. . . . . . . . . . . . . . . . .
Relation of Chemical Composition to Presence of Bacteria. . .
Discussion.
...............
. . . .
. . . . . . .
Summary
. . . .
................
. . . . . . . .
References. . . . . . . . . . . .
. . . . . . . .
. . . . .
. . . . .
v
1
4
6
7
10
10
10
12
12
13
16
19
19
19
40
42
62
64
68
74
75
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Figure
FIGURES
Page
1.
Map of West t~orhead, Decker, and Colstrip areas
of the Fort Union coal region. . . . . . . . . .
Map of Bear Creek study area showing overburden
core drilling sites. . . . . . . . . . . . . . .
.......
9
.......
8
2.
3.
Al ga 1 growth curves of Sel,ena8trum aapriaoPnuturn
Printz, comparing six methods of biomass monitoring.
.....
41
4.
Algal growth studies of Sel,ena8trum aapriaoPnuturn
Printz, determining the influence of five factors on
algal growth, as measured by in vivo fluorescence method. . . 43
Algal control growth curves of SeZena8trum aapriaoPnuturn
Printz, used with different leachate bioassays as determined
by in vivo fluorescence measurements. . . . . . . . . . . . . 44
5.
vi
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TABLES
Table
1. Autotrophic growth media. . . . . .
. . . . .
.........
2.
Percent of thiosulfate oxidized and resulting pH
values for thiobacilli. . . . . . . . . . . . .
........
3.
Content of the leach flasks for both static and
shaking conditions. . . . . . . . . . . . . . . . . . .
. . . .
4. Analysis of core sample DH75-102. . . . . . .
5. Analysis of core sample DH75-103. .
. . . . .
. . . .
.....
.....
. . . .
6. Analysis of core sample DH75-104. .
..............
7.
Analysis of core sample DH75-106(A) .
.............
8. Analysis of core sample DH75-106(B) .
9. Analysis of core sample DH76-108.
.............
. . . .
. . . . . .
. . . . .
10. Analysis of core sample DH75-109. .
11. Analysis of core sample DH76-111. .
..............
.........
. . . . .
12.
Percent of oxidized thiosulfate and ferrous-iron, and
resulting pH from enrichment cultures of core sample
D H7 5 -102 . . . . . . . . . . . . . . . . . . . . . . .
.....
13.
Percent of oxidized thiosulfate and ferrous-iron, and
resulting pH from enrichment cultures of core sample
DH75-103. . . . . . . . . . . . . . . . . . . . . . .
. . . . .
14.
Percent of oxidized thiosulfate and ferrous-iron, and
resulting pH from enrichment. cultures of core sample
D H7 5 -104 . . . . . . . . . . . . . . . . . . . . . . .
. . . . .
15.
Percent of oxidized thiosulfate and ferrous-iron, and
resulting pH from enrichment cultures of core sample
DH75-106{A) . . . . . . . . . . . . . . . . . . . . .
. . . . .
vii
Page
11
14
15
20
21
22
23
24
25
26
27
28
29
31
33
-------
Table
16.
17.
Percent of oxidized thiosulfate and ferrous-iron and
resulting pH from enrichment cultures of core sa~ple
DH75-106( B) . . . . . . . . . . . . . . . . . . . . .
. . . . .
Percent of oxidized thiosulfate and ferrous-iron and
resulting pH from enrichment cultures of core sa~ple
DH7 6-108. . . . . . . . . . . . . . . . . . . . . . .
.....
18.
Percent of oxidized thiosulfate and ferrous-iron, and
resulting pH from enrichment cultures of core sample
o H7 5 -1 09. . . . . . . . . . . . . . . . . . . . . . .
. . . . .
19.
Percent of oxidized thiosulfate and ferrous-iron, and
resulting pH from enrichment cultures of core sample
D H7 6 -111 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20.
Core and leachate analyses and algal bioassays, core
s amp 1 e DH7 5 -1 02 . . . . . . . . . . . . . . . . . . . . . . . .
21.
Core and leachate analyses and algal bioassays, core
sample DH75-103 . . . . . . . . . . . . . . . . . . .
. . . . .
22.
Core and leachate analyses and algal bioassays, core
sample DH75-104 . . . . . . . . . . . . . . . . . . .
Core and leachate analyses and algal bioassays, core
sample DH75-106(A). . . . . . . . . . . . . . . . . .
. . . . .
. . . . .
23.
24.
Core and leachate analyses and algal bioassays, core
sample DH75-106(B). . . . . . . . . . . . . . . . . . . . . . .
25.
Core and leachate analyses and algal bioassays, core
sampl e DH76-108 . . . . . . . . . . . . . . . . . . .
Core and leachate analyses and algal bioassays, core
sample DH75-109 . . . . . . . . . . . . . . . . . . .
. . . . .
.....
26.
27.
Core and leachate analyses and algal bioassays, core
sample DH76-111 . . . . . . . . . . . . . . . . . . . .
Results of algal bioassays of leachates of core samples
following pH adjustment to pH 8.0, percent stimulation
or inhibition. . . . . . . . . . . . . . . . . . . . .
. . . .
. . . .
28.
29.
Chemical analysis of overburden core samples in
relation to presence of bacteria which oxidize iron
and sulfur compounds. . . . . . . . . . . . . . . .
......
viii
Page
34
36
37
39
45
47
49
51
53
56
58
59
65
66
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ACKNOWLEDGEMENTS
We thank George M. Pike, District Chief, and Donard L. Coffin,
Supervisory Hydrologist, both of the U.S. Department of the Interior
Geological Survey, Water Resources Division, Helena, Montana, for
assistance in initiating this project and for supplying the overburden
cores, maps, and logs.
The research was funded both by the U.S. Geological Survey and by
the U.S. Environmental Protection Agency, Environmental Research Laboratory-
Duluth, Minnesota, Research Grant No. R803950, awarded to Natural Resource
Ecology Laboratory, Colorado State University. and Fisheries Bioassay
Laboratory. Montana State University.
ix
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SECTION I
INTRODUCTION
The replacement of a deep coal deposit and overburden by crushed over-
burden (spoils) in the Fort Union coal area means the replacement of a known
aquifer by one with unknown characteristics. Consequently it is of interest
to determine the changes in water quality that may be expected to occur from
the leaching that will inevitably take place when water moves through spoils.
Crushed sedimentary rock exposed to air and water is subject to bacterial
leaching as well as to chemical leaching. This combination of chemical and
bacterial leaching usually results in changes in total dissolved solids, in
salt content, hardness, and conductivity as well as in the concentration of
many metal and nonmetal elements. Bacterial leaching is most pronounced in
disturbed sedimentary strata when these contain appreciable amounts of
sulfides. The Fort Union coal is rated as low in sulfur. However, there are
local accumulations of pyrite such that coal blending is occasionally necessary
to meet the low sulfur requirement before shipping. The overburden contains
highly variable but usually small amounts of sulfides. In toto, the amounts
of sulfides are not great enough to presuppose an acid problem. Sulfides are
frequent enough to suggest that bacterial leaching will be influenced by their
presence.
The solubilization of metals from mining spoils, including coal mine
spoils, has been well documented by Galbraith et al. 1972, Silverman and
Ehrlich 1964, Tuovinen and Kelly 1974, and Fjerdingstad et al. 1976. The
metals, as sulfides, will oxidize upon exposure to oxygen resulting in the
formation of the corresponding metal sulfates, by the following reaction
(Bosecker et al 1978; Fjerdingstad et al 1976; Razzell and Trussell 1963;
Silverman and Ehrlich 1964; and Temple and Delchamps 1953).
MeS + 202 = MeS04
The presence of pyrite (iron disulfide, FeS2) in spoils causes the most
vigorous solubilization of metals, as a consequence of bacterial production
of ferrous, ferric, sulfate and hydrogen ions from the pyritic material
(Singer and Stumm 1970; Temple and Delchamps 1953; and Temple and Koehler
1954). The ferric ion, produced through bacterial action, is a potent
chemical oxidant, resulting in the solubilization of minerals (Singer and
Stumm 1970; Temple and Delchamps 1953). Members of the bacterial genus
ThiobaciZZus catalyze the solubilization of metal sulfides due to the general
ability of the genus to oxidize reduced sulfur compounds and to the specific
ability of the species, ThiobaciZZus ferrooxidans, also to oxidize reduced
1
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ferrous iron ion (Silverman and Ehrlich 1964; Singer and Stumm 1970; Temple
and De~champs 1953; and Temple and Koehler 1954). Temple and Koehler (1954)
r~cogn~zed that ~he overall reaction sequence was a mixture of chemical and
blologlcal reactlons which they expressed as:
Initial chemical reaction:
FeS2 + 3~ O2 + H20 = FeS04 + H2S04
Bacterial reaction carried out by T. ferrooxidans:
2 FeS04 + ~ O2 + H2S04 = Fe2(S04)3 + H20
Subsequent chemical reactions:
Fe2(S04)3 + FeS2 = 3 FeS04 + 2 S
Bacterial reaction carried out by T. thiooxidans:
+ =
S + 1~ O2 + H20 = 2 H + S04
According to Silverman (1967) and Beck and Brown (1968) the biological
oxidation of pyrite is thought to proceed by two mechanisms concurrently: a
direct contact mechanism which requires physical contact between bacteria
and pyrite particles and an indirect contact mechanism according to which
the bacteria oxidize ferrous ions to the ferric state, thereby regenerating
the ferric ions required for chemical oxidation of pyrite. Singer and Stumm
(1970) and Bosecker et al. (1978) postulate that the oxidation of metal sul-
fides in the presence of pyritic material is mainly restricted to the chemical
oxidation of sulfides via ferric iron. They also postulate that the activity
of chemoautotrophic bacteria is mainly restricted to the reoxidation of
chemically reduced ferric iron. Bryner and Anderson (1957) and Brierley (1978)
observed the bacterial oxidation of a metal sulfide, molybdenite (MOS2)' in
the absence of pyrite. From these references, it could be assumed that
oxidation of metal sulfides by chemoautotrophic bacteria will occur with or
without pyrite, but that the rate of oxidation will be more rapid when
associated with pyrite. Other bacteria and fungi, such as Bacillus species
and penicillium, have been shown to aid in the solubilization of metals,
through the production of organic acids and some unidentified compounds
(Tuovinen and Kelly 1974). However, the contribution of heterotrophic micro-
organisms in metal solubilization may be limited, due to low leaching yields,
whereas the participation of Thiobacillus species in metal solubilization
has been widely accepted and exploited (Tuovinen and Kelly 1974).
Visible halos are frequently seen surrounding metal ore deposits. These
halos consist of dissolved and reprecipitated minerals, often acid salts, and
are formed by the chemical reaction of bacterially produced sulfuric acid
with carbonates found in the ore or overburden (Silverman and Ehrlich 1964).
These halos are common in Fort Union coal strip mine reclaimed spoils.
During rainy periods, these salts are dissolved and eventually enter the
ground water.
This study on overburden leaching was initiated by the U.S. Geological
Survey with cores supplied by them and was continued with support from the
EPA. The EPA. recommended Selenastrum algal assay test was used to determine
toxicity of leachates.
2
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As coal mining expands with the current and future energy demand, a more
complete understanding of alkaline and acid mine drainage from coal mine
spoils is required. This project studied coal mine spoils from eastern
Montana which is characterized by alkaline mine drainage. The method of this
project was to leach core samples under conditions in which bacterial leaching
might occur and to examine the leachates for,toxicity by the algal assay.
Analyses for Pb were also made in the cores and their leachates, since this
element is both a toxic material and a possible indication of autotrophic
leaching. The purpose of these procedures was to provide a spot check on
possible potential water problems which might be caused by bacterially
assisted leaching of disturbed overburden.
3
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SECTION II
CONCLUSIONS
The following conclusions are based on a limited study of individual
strata from coal overburden cores. The core samples available to us were
grab samples, chosen by an experienced geologist after core logging but
without the benefit of mineralogical or chemical analysis. Our conclusions
are indicative, but should not be considered comprehensive.
1. leachates from the individual strata which we examined varied both
in pH and their toxicity to algae from causes other than acidity.
2. Most of the strata tested produced leachates that were either basic,
neutral, or weakly acidic in reaction. These leachates would not consitute
an acid problem under field conditions. A few of the strata we tested pro-
duced strongly acid leachates. These acid-forming strata amount to a rela-
tively very small part of the total overburden mass in any core.
3. The lead content of the samples which were leached did not correlate
with the lead content of the leachates. Since a positive correlation is
expected when lead and pyrite occur together, we conclude that there is no
such association in the samples tested. Whether any other potentially toxic
elements were present in the few cases of acid leachates is a question that
was not examined.
4. Several leachates proved to be toxic according to the algal bioassay.
A few of these were toxic because of their low pH. The toxicity of the others
was confirmed by repeated experiments but the chemical basis for their toxi-
city was not determined.
5. The biological leaching procedure used in this study could be used
to identify both acid-forming strata and toxic leachate-forming strata.
6. An extrapolation of these results to the coal field reclamation
situation leads to the conclusion that both acid leachates and toxic leachates
would be rarely encountered. This study was designed as an exploratory spot
check. It is therefore impossible to extrapolate from it to quantitative
conclusions. The overburden at the sites where our cores were taken consists
of roughly horizontal strata which individually vary from less that a few
centimeters to several meters in thickness, but which in total add up to
between 28 and 93 meters of overburden. It is well known that any given
stratum undergoes changes in chemical and mineralogical make-up over the
horizontal distance of a coal field. For an area as extensive as the Fort
4
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Union coal, these changes should be major ones. Any stratum might vary greatly
in thickness, perhaps disappearing entirely in some localities, and having
different chemical composition depending on the local sedimentary conditions
of the basin at the time of deposition. Our spot check of selected samples
from a few cores in a small part of this coal district agrees with the general
opinion that Fort Union coal has for the most part an innocuous overburden.
This spot check has also pointed out that some overburden strata, at some sites,
are potentially toxic and that others are potentially acid-forming. A quan-
titative prediction about these features in the coal field as a whole requires
considerable guesswork, when spot check samples are the basis for that pre-
diction.
7. With due regard to the reservations expressed in Conclusion 6, we
conclude that acid formation would be a problem only in the circumstance when
a potentially acid-forming stratum is so placed during spoil reclamation that
it drains directly into a surface stream. The physical effect of heavy equip-
ment during overburden removal, stockpiling, and resurfacing is to break up
the relatively soft sedimentary rocks and to produce a haphazard but real
mixing of strata. This minimizes the acid potential, particularly since the
greater mass of overburden consists of strata which do not produce acid and
which often contain carbonate minerals capable of neutralizing acidity.
8. The same reasoning suggests the conclusion that toxicity due to
materials other than acid would be rare in these coal fields. However, the
existence of toxic leachates from a few samples among those tested prompts
the conclusion that there may be a real risk under some circumstances. Our
spot check has not adequately established the degree of this risk. We know
that some companies engaged in surface coal mining in the west segregate
certain strata on the basis of their chemical analyses and reserve these strata
for special precautionary handling during reclamation. From our results, we
conclude that toxicity testing by a bioassay is a useful adjunct to chemical
analysis for identifying problem strata.
9. Although not a part of this study, other observations by us in eastern
Montana coal fields have demonstrated local acid spots in surface spoils which
have been reclaimed by the best current practice. In these cases, the acid
leachate is neutralized by carbonate minerals in the immediate vicinity of the
pyrite and does not enter the ground water supply. These acid spots are recog-
nizable as halos of precipitated iron oxide surrounding masses of nodular
pyrite, which, when tested, prove to be highly acidic. The only apparent
effect is the absence of plant roots in the acid zone. Pyrite in this reclaimed
coal field consists of nodular masses of pyrite mixed with other minerals
including carbonates, and also as large masses of museum grade cubical crystals
of apparently pure pyrite. Both types oxidize slowly. In contrast, the acid-
forming samples which we tested in this study do not contain any visible pyrite
but do oxidize rapidly. This is an example of the expected variability in
stratum composition referred to in Conclusion 6.
5
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SECTION III
RECOMMENDATIONS
1. Individual strata from the overburden should be tested at several
sites within an operating coal field prior to overburden removal. Recom-
mendations based on visual examination of cores by a qualified geologist or
geochemist would minimize the number of samples required for analysis.
Strata with a high carbonate content need not be tested for acid formation.
However, acid formation potential tests should not be restricted to samples
with visible pyrite.
2. Leaching tests should be used to determine acid potential. These
tests are more reliable than the chemical analyses sometimes recommended
because of the great difference in the rate of oxidation of different pyrite
deposits. The leaching study could be quantitative as well as qualitative.
3. Leaching tests should be used to determine potential toxicity of
individual strata. In addition to pointing out potentially toxic strata,
leaching tests might determine whether a toxic element in a stratum, found
by chemical analysis of the stratum, is actually leached.
4. Strata found to be either potentially acid-forming or potentially
toxic should be segregated during overburden removal and should be deeply
buried in a region with minimal underground water flow and strongly com-
pacted during reclamation, to minimize leaching.
5. The cause of the unexplained toxicities found in this study should
be ascertained.
6
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SECTION IV
DESCRIPTION OF STUDY AREA
The study area was the Bear Creek coal field in the West Moorhead coal
deposit of the Fort Union coal formation in eastern Montana (Fig. 1). Over-
burden cores were obtained from an area which, to date, is not being mined
(Fig. 2). One overburden sample was taken from a revegetation study site,
1969-11, at Colstrip, Montana, which is also part of the Fort Union coal
formation (Fig. 1). The sample was obtained from an exposed chunk of coal in
the overburden which was surrounded by an acid salt halo. These halos develop
from the weathering of the large numbers of nodular pyrite masses which are
spread over several hectares of reclaimed spoil.
7
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ex>
Fig.
I
r- --.. ---- -- -
I -- ------------
.
)
'\
'7
"
t~
,
"
~ r-
\.r-I'r-"I'\
I WYOMING
,
G rea t
Fall. I
M
Bozeman I
1.
o
N
T
A
f2J
Fort
Coal
Union
Reolon
Map of West Moorhead, Decker, and Colstrip areas of the Fort Union coal
(Adapted from Van Voast and Hedges 1975.)
region.
-------
R 45 E
/ I
/ /
( (
\ - -po..
- ---
Contour interval 100'
Water - table contour interval 20'
Direction of water movement
BEAR CREEK STUDY AREA
(Adapted from USGS hydroloQic
mop.)
-
@
o
o
Well or test hole
1000 2000 3000 feet
300 600 900 meters
Fi g. 2.
Map of Bear Creek study area showing overburden core drilling sites.
9
-------
SECTION V
MATERIALS AND METHODS
Media
Water utilized. The water utilized was either double-distilled water
or reagent grade water that had been processed by a Milli-Q water system
(Millipore Corporation, Bedford, Massachusetts) following single distillation
and was stored in Pyrex glass.
Autotro hic media. The autotrophic media were described by Hutchinson
et ale 1965,1966,1967), Silverman and Lundgren (1959), and Manning (1975).
These media utilized thiosulfate or ferrous-iron as their energy sources,
and are listed in Table 1.
The ISP medium was utilized only as a solid medium, while other media
were utilized as both liquid and solid media. To solidify the medium,
"Ionager" no. 2 (Colab Laboratories, Chicago Heights, Illinois) was added at
a concentration of 1.5%.
Leachate medium. The basil salt medium of Brierley and Brierley (1973)
was used in the leaching experiments. This medium was used with glucose
(0.1% w/v) or without glucose. The medium pH was adjusted to the measured
pH of the core sample being leached.
Algal Bioassay Medium. The medium (AAP) in the algal bioassays was
prepared as described by the U.S. Environmental Protection Agency (1971)
Preparation of Glassware
All glassware, except pipettes, was machine washed and air dried. Items
that were acid washed were soaked in 3 N HCl for a minimum of thirty minutes,
rinsed six times with tap water followed by six rinses with double distilled
water or Milli-Q reagent grade water, and air dried. Glassware sterilized
by autoclaving was either covered with aluminum foil or stoppered with
gauzed cotton plugs and processeq for 15 minutes at 15 pounds pressure.
Pipettes were soaked in chromic acid for 30 minutes, rinsed at least
fifteen times with tap water and air dried. Pipettes and glass petri dishes
were placed in metal cans or boxes and oven sterilized at 177°C for 3 hours.
10
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Table 1. Autotrophic growth media.
Reference
Acid
thiosulfate
Media
neutral
thi osulfate
Ferrous-iron
Hutchinson et al. (1965)
Silverman and Lundgren (1959)
S6
S5
Fe
9K
Manning (1975)
ISP
11
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Sampling
Overburden grab samples were obtained from eight test holes of the Bear
Creek coal area of the West Moorhead coal field of the Fort Union coal
deposit in eastern Montana by the U.S. Geological Survey. Each of the eight
test hole cores was accompanied by a geological log. Each sample consisted
of a portion of the test hole core, between known depths, and was placed in
plastic bags for transport. The samples were stored in plastic bags at
room temperature in the dark.
A sample was obtained from a revegetation study site (1969-11) at Colstrip,
Montana. The sample was taken from an exposed area which exhibited evidence
of iron oxidation in close association with coal distributed in the over-
burden. This sample was stored in a plastic bag at room temperature.
Sample Analysis
Each sample was visually inspected for evidence of mineralization. Core
samples were then crushed in a mortar and pestle, analyzed for pH, conductivity,
and lead, and cultured for chemoautotrophic bacteria.
pH measurement. For pH measurement, 10 g of ground sample were mixed
with 20 ml of double distilled water and allowed to stand for four hours before
making the pH reading with a Radiometer model 25 pH meter. Readings were
stable at this time.
Conductivity measurement. Conductivity was measured by adding 10 g of
ground sample to 20 ml of double distilled water of known conductivity, and
allowing the suspension to stand for 4 hr before measuring. A Lab-Line Lecto
model MC-1 Marke IV ohm meter was used. The listed values are therefore rela-
tive figures representing the conductivity of 20 ml of water which has been
exposed to contact with 10 9 of crushed sample. These values should not be
considered to represent the specific values of leach water. The conductivity
was recorded as ~mhos/cm at 25°C.
Lead measurement. Each sample was extracted by adding 3 ml of concentrated
HCl and 1 ml of concentrated HN03 to 1 g of ground sample in an acid washed
screw capped tube. The mixture was boiled for 1-1/2 minutes prior to the
addition of 3 ml of double distilled water, followed by boiling for another
1-1/2 minutes. The extract was cooled and filtered through Whatman no. 4 fil-
ter paper into an acid washed 10 ml volumetric flask. The flasks were filled
to volume with double distilled water. The extracts were mfxed and then
stored in acid washed screw capped tubes at 4°C until analyzed. Lead was
measured by atomic absorption spectrophotometry, using an Instrumentation Lab-
oratory. Inc. model 151 atomic absorption/emission spectrophotometer. The
lead concentration was reported as ~g per 9 core sample.
Bacterial enrichment. Enrichment cultures for the chemoautotrophic
bacteria, thiobacilli, were attempted with each sample. One qram of ground
sample was added to each of three 125 ml erlenmeyer flasks, each flask con-
tained one of 3 media, 56, 55, or Fe. These flasks were incubated statically
at 28°C in the dark. After two weeks incubation, 0.5 ml from each flask was
subcultured into 5 ml of fresh medium in capped tubes. After one month
12
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incubation, pH and thiosulfate or ferrous-iron concentration were measured on
each of the original enrichment flasks (Skoog and West 1963; Sorbo 1957). The
amount of both thiosulfate and ferrous-iron oxidized was reported as a percent
of the uninocu1ated control incubated for the same one month period. The pres-
ence of chemoautotrophic bacteria was determined from the resulting percentages
and from elemental sulfur flocculation and from turbidity of the medium or
from visible iron oxidation. The liquid cultures showing growth were trans-
ferred to fresh liquid medium and also plated on solid medium. When growth
in the liquid medium transfers did not result, transfers were made to liquid
medium (S6, S5, or Fe) which had been supplemented with yeast extract (0.05%,
w/v), cysteine (10 mg/100 m1; J. A. Brierley, private communication), gluta-
thione (10 mg/100 m1), or IM-MF additives (0.1%; Stuart et a1. 1977). Liquid
cultures were subsequently transferred weekly. Representative colonies were
selected from the plates at weekly intervals and restreaked for purification
of the culture. Strains were purified by at least three single colony isola-
tions (Hutchinson et a1. 1965). Pure cultures were characterized to species
according to the scheme of Hutchinson et a1. (1969) for thiobaci11i taxonomy.
The pH and percent thiosulfate oxidized values for thiobaci11i according to
the diagnostic tests of Hutchinson et a1. (1969) are illustrated in Table 2.
The sample from the revegetation study site was cultured for ferrous-iron
oxidizing bacteria in Fe medium. The culture was subsequently transferred to
9K medium and 9K medium plus yeast extract (0.05%, w/v), cysteine (10 nlg/l00 m1).
glutathione (10 mg/l00 m1), or IM-MF additives (0.1%). Iron oxidation and
final pH were measured after incubation for 18 days.
Leaching Studies
Core sample selection. Samples were selected which exhibited a wide
range of pH values, conductivity values, and lead contents.
Samples to be leached were ground and sized to
Autotrophic inoculum. Autotrophic bacteria, both sulfur and iron oxi-
dizing bacteria, were obtained from various sources. Sulfur oxidizing
bacteria were isolated from the Artist's Paint Pot area and Geyser Springs,
Yellowstone National Park. Iron oxidizing bacteria, isolated from the
settling pond of the Decker Coal Mine, Decker, Montana, were supplied by
Dr. Greg Olson. These cultures were maintained separately, and mixed prior
to inoculation of the leach flasks.
Soil inoculum. A general soil inoculum was obtained from greenhouse
pots immediately prior to the inoculation of the leach flasks.
Experimental leaching design. Leaching was performed in er1enmeyer flasks,
under both static and shaken conditions. The shaker was a New Brunswick model
VS gyrotory shaker (New Brunswick Scientific Co~pany, New 8runswick, New Jersey)
operated at a speed of 180 revolutions per minute. The flask size, the
medium volume, core sample weight, and inoculum volume or weight differed for
the two conditions, as shown in Table 3. Six flasks were used for each
condition for each core sample. Each of the six flasks contained core sample.
13
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Table 2.
Percent of thiosulfate oxidized and resulting pH values
for thiobacilli. [According to the diagnostic tests of
Hutchinson et al. (1969).]~
Percent
thiosulfate Resulting
ThiobaaiLLus species oxidized pH
ThiobaaiLLus noveLLus <30% 6.6-5.0
ThiobaaiLLus denitrifiaans <90% 6.6-5.0
ThiobaaiLLus thioparus >90% 6.6-3.5
ThiobaaiLLus neapoLitanus >90% 3.5-2.8
ThiobaaiLLus thiooxidans >90% <2.0
ThiobaaiLLus ferrooxidans >90% <2.0
ThiobaaiLLus intermedius >90% <2.8->2.0
~Values determined from cultures grown in 56 or 55 medium for a
period of 28 days.
14
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Table 3. Content of the leach flasks for both static and shaking conditions.
Erlenmeyer Core sample t1ed i urn Inoculum Size
flask volume weight volume autotrophic soil
Cond it ion (ml) (g) (ml) (ml) (g)
Static 250 5 150 1.0 5
Shaking 125 2.5 75 0.5 2.5
15
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Three of the six flasks contained the leach medium without glucose added,
while the others contained the leach medium with glucose added. The inoculum
was the same for both sets of three flasks; one flask was an uninoculated
sample control, one flask was inoculated with the mixture of autotrophic
bacteria, and one flask was inoculated with soil from the greenhouse pots.
The experiment was incubated for 30 days at room temperature in the dark.
Handling and analyses of leachates. Following the leaching period, a
15 to 20 ml portion of each leachate was removed to obtain the pH reading and
lead content. Prior to lead analysis the samples were filtered through What-
man no. 4 filter paper into acid washed screw capped tubes and stored at 4°C.
The remaining leachate was used in the algal bioassays. This portion
of the leachate was prefiltered (Millipore, AP25) followed by filtering through
0.45 ~m membrane filter (Millipore, type HAWP) into sterile glassware. The
leachate was stored in acid washed containers, either plastic bottles or glass
tubes, at 4°C until the bioassay was performed.
Lead is reported as ~g/g of core.
Algal Bioassays
Organism utilized. A culture of Selenastrum aapriaornutum Printz was
obtained from the Environmental Protection Agency, Corvallis, Oregon.
Culture maintenance. A culture of Selenastrum aapriaornutum Printz was
maintained on AAP medium (U.S. EPA 1971) by transfer of 5 ml of culture to
100 ml of fresh medium in 250 ml erlenmeyer flasks. The recommended routine
stock culture transfer schedule of weekly transfers (U.S. EPA 1971) was
initially decreased to 6 days, but after further experiments transfer every
2 days was found to give more reproducible assays.
Test conditions. All flasks, either for maintenance or bioassay, were
incubated at room temperature under continuous cool-white fluorescent lighting
at 400 ft-c (U.S. EPA 1971). The flasks were continuously shaken at approxi-
mately 100 oscillations per minute on aGIO gyrotory shaker (New Brunswick
Scientific Company, Inc.). Growth curve studies were performed with 10 repli-
cate 500 ml erlenmeyer flasks containing 200 ml of AAP medium. All other
bioassays were performed in 250 ml erlenmeyer flasks containing 100 ml of
AAP medium.
Preparation of inoculum. Initially, cells from the stock culture were
prepared by washing as described by the U.S. Environmental Protection Agency
(1971). Centrifugation was performed at 10,000 rpm for 10 minutes using a
$orval superspeed RC2-B automatic refrigerated centrifuge. After some experi-
mentation, the inoculum was taken directly from the flasks, without washing
and centrifuging.
Amount of inoculum. The starting cell concentration in the test flasks
was 10j cells per ml (U.S. EPA 1971). The cell concentration (X) was deter-
mined from the fluorometer reading (Y) by the following equation (Fitzgerald
and Uttormark 1974).
16
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X
103 cells/ml
=
Y
15.4 relative fluorescence units
Preparation of glassware. Glassware was acid washed and sterilized as
previously described. Test flasks were stoppered with gauzed cotton plugs.
Biomass monitoring for growth curve studies. Several methods were used
to determine the biomass during the growth curve studies; these methods are
subsequently described.
Turbidity. In vivo optical density was determined using a Varian Techtron
model 635 spectrophotometer at 750 nm (U.S. EPA 1971) with a one centimeter
path length in the cuvette.
Fluorescence. Fluorescence was used as one means of measuring chlorophyll,
with both in vivo and extracted suspensions (U.S. EPA 1971). The extraction
methods described by Yentsch and Menzel (1963) were followed with the following
substitutions: 0.45 ~m cellulose acetate membrane filter (Millipore) for glass
fiber filter, sodium bicarbonate (15 mg/l) for magnesium carbonate (1 g/100 ml),
5 ml of 90% acetone for 2 ml of 90% acetone added to the grinding tube, and
after grinding for 1 to 2 minutes, samples were frozen overnight prior to
centrifuging instead of allowing centrifuged samples to stand for 1 to 2 hours.
The tissue grinder was a Lightnin model L mixer (Mixer Equipment Company, Inc.,
Rochester, New York). Fluorescence was determined using a G. K. Turner
Associates model no. 111 fluorometer (Palo Alto, California). Chlorophyll
and phaeophytin were measured by reading the initial fluorescence (FS)' and
the fluorescence (F ) after the addition of 2 drops of 2 N HC1. Chl rophyll a
(Fchl) was calculat~d from the formula of Yentsch and Menzel (1963).
FChl = 1.77 (Fo - Fa)
Chlorophyll a was used in the plotting of the extracted chlorophyll data.
Absorbance. Chlorophyll was determined by absorbance using the Varian
Techtron spectrophotometer at 665 nm and 750 nm (U.S. EPA 1971; Yentsch and
Menzel 1963). Extraction and acidification methods were performed as for
chlorophyll a fluorescence. Chlorophyll a and phaeopigment a were calculated
according to the formula in Weber (1973) and Standard Methods (APHA et ale 1976).
26.7(665b - 665a) E
chlorophyll a = VL
26.7[1.7(665b) - 665aJ E
phaeopigment a = VL
665a =
665b =
E =
V =
L =
absorbance after acidification
absorbance prior to acidification
volume (ml) of 90% acetone added
volume (ml) of extract filtered
path length (cm) of cuvette, 1 cm
17
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Cell count. Direct cell count was determined using a Petroff-Hausser
bacterial counter (C.A. Hausser and Son, Philadelphia, Pennsylvania).
Dry weight. Dry weight and ash-free weight were determined following
the methods of Weber (1973). Five ml of sample were pipetted into the tared
porcelain crucible.
Biomass monitoring for bioassays. In vivo chlorophyll a fluorescence was
used to monitor the biomas.s in the bioassay studies. The data were reported
as percent inhibition, indicated by a negative value, or stimulation, indicated
by a positive value, as compared to the maximum standing crop of algal control
flasks. The values were calculated by the following formula.
x = 100 (A - B)
B
X = tabulated value, percent
A = leachate bioassay fluorescence reading
B = algal control fluorescence reading at maximum standing crop
Maximum standing crop is defined as the maximum algal biomass reached during
incubation (U.S. EPA 1971).
Algal growth studies. The growth of SeZenastrum aapriaornutum Printz
as influences by either pH, glassware cleanliness, inoculum size, inoculum
age, or inoculum washing was followed by fluorescence readings.
Medium pH. The pH of the AAP medium was adjusted with a sodium acetate-
acetic acid buffer (Meynell and Meynell 1970) to five pH values, 3.6, 4.2,
4.6, 5.0, and 5.4.
Glassware acid washing. Glassware was washed with boiling chromic acid
which was swirled to coat the glassware surface, rinsed 3 times with tap water,
rinsed 3 times with a 3:1 concentrated HCl:concentrated HN03 solution, and
rinsed 6 times with both tap water and double distilled water.
Inoculum size. The glassware acid washing study was run in conjunction
with a study in which the inoculum size was doubled from 103 cells per ml
to 2 x 103 cells per ml.
Inoculum age. The inoculum age was varied from the usual six-day-old
inoculum to two- and four-day-old inocula.
Inoculum washing. Inoculum washing was examined by inoculating flasks
with cells washed in sodium bicapbonate (15 mg/l) and with cells unwashed.
Algal bioassays of leachates. Leachate bioassays were performed uSing
10 ml of filtered leachate, 90 ml of AAP medium, and a volume of cells as
inoculum so that the test flasks contained 103 cells per ml. Leachates which
produced acid inhibition in the algal bioassays were reassayed after raising
the pH to pH 8.0 with IN NaOH. Fluorescence values are net values of medium
plus leachate plus alga over medium plus leachate without alga.
18
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SECTION VI
RESULTS
Sample Analysis
The diversity of the core samples can be observed from the analyses pre-
sented in Tables 4-11. Each core sample was composed of some combination of
shale, sandstone, siltstone, coal, or clay. There was visual evidence of
metal mineralization in some samples, exemplified by the presence of iron
oxidized portions and salt crystals. Some layers of the overburden cores
were composed entirely of coal, as seen in Tables 4, 5, 7, and 10, while each
of the cores had coal as a part of some of their samples. The pH of most
samples was above 7.0, and often as high as pH 8.~, with the highest pH value
being pH 9.65 (Table 10). The majority of the samples containing coal had pH
values of less than pH 5.54, but some samples with no coal also had low pH
values. The sample with the lowest pH value, pH 2.31 (Table 11), contained
coal along with carbonaceous shale. Lead content of the samples had a maxi-
mum of 57.0 ~g per g of sample but the majority of samples had less than
20 ~g/g of sample (Tables 5 and 11). The amount of lead in the sample did
not appear to be related to any of the other analyses. Conductivity values
ranged from the 162 ~mhos/cm to 9232 ~mhos/cm at 25C (Table 11), but most
samples had conductivities below 3000 ~mhos/cm. Conductivity values were
not consistently related to either the physical composition or to the other
chemical components measured, although samples with pH values less than pH
5.73 [except for DH75-106(B) sample depth 270.5-280 ft (Table 8)J also had
higher conductivities than most other samples. These high conductivities
ranged from 3422 ~mhos/cm at 25C to the highest conductivity of 9232 ~mhos/cm
at 25C. Conductivities equally great also occurred with samples having pH
values higher that pH 5.73.
Enrichment Cultures
Enrichment cultures for thiosulfate oxidizing bacteria in S6 and S5 media
and for ferrous-iron oxidizing bacteria in Fe medium, were incubated for one
month. The amount of thiosulfate or iron oxidation and the resulting pH were
determined (Tables 12-19). The presence of bacteria was substantiated in many
cases by wet mount microscopic examination, by observation of sulfur floccu-
lation on the surface of the medium, or by turbidity. As Table 3 demonstrates,
the different species of thiosulfate oxidizing bacteria exist over a wide pH
range and oxidize variable percentages of thiosulfate. The S6 medium produced
more cultures which oxidized 90% or more of the thiosulfate than did the 55
medium. The final pH values from 90% oxidation of thiosulfate in 56 medium
varied from pH 6.45 to pH 2.45, which encompassed three groups of ThiobaciZZus
species. The two groups which oxidize less than 90% of the thiosulfate were
19
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Table 4. Analysis of core sample DH75-102.
Depth Pb Conductivity
(ft) Description pH (~g/g) (~mho/cm,25 C)
,
0- 10 Sandy clay; yellow, Fe-oxidized 8.15 6.8 3449
spots
1 0- 20 Sandy clay 8.64 4.3 3359
20- 30 Clay; yellow, Fe-oxidized 8.43 7.5 1001
pebbles
30- 40 Sandy clay, carboniferous 8.49 8.2 829
shale; yellow, Fe-oxidized
pebbles
40- 50 Coal 5.47 2.7 4929
50- 60 Shale and sandstone; 8.46 14.4 3059
carboniferous specks
60- 70 Siltstone (light grey) 8.24 5.6 849
70- 80 Shale and siltstone 8.63 4.8 1358
80- 90 Shale; carboniferous fragments 7.29 2.8 1429
90-100 Si ltstone; carbonaceous spots 7.41 4.8 1441
1 00- 11 0 Sil tstone; carboniferous specks 7.48 12.4 2206
20
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Table 5. Analysis of core sample DH75-103.
Depth Pb Conductivity
(ft) Description pH ()Jg/g) ()Jmho/cm, 25 C)
0- 10 Sandy shale; Fe-oxidized spots 8.43 19.3 3133
10- 20 Sandstone; carbonaceous streaks 8.32 23.8 3703
20- 30 Shale; orange (Fe-oxidized) 8.14 13.2 1714
spots
30- 40 Carbonaceous clay; coal 7.69 15.1 1803
40- 50 layered coal 6.51 1.9 1032
50- 60 Coal 5.54 6.3 3922
60- 70 Shale 8.65 15.6 678
70- 80 Shale and sandy siltstone 7.42 7.6 1210
80- 90 Shale 8.51 10.6 986
90-100 Sandstone 8.67 4.7 772
1 00- 11 0 Shaley sandstone; carbonaceous 8.49 49.1 1060
streaks
110-120 Shale with carbonaceous 6.67 21.8 1830
specks and coal
120- 130 Sandy shale 6.23 24.3 2826
130-140 Sandstone and shale 8.59 4.7 1062
140- 150 Coal and sandstone 8.92 10.3 892
150-160 Shale; carbonaceous 1 ayers 8.65 14.5 1485
160-170 Sandstone with Fe-oxidized 7.91 8.6 1775
spots; shale with carbonaceous
s trea ks
170-180 Silty clay 8.71 7.3 1191
180-188 Silty clay; carbonaceous spots 8.99 6.9 918
21
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Table 6. Analysis of core sample DH75-104.
Conductivity
Depth Pb (Ilmho/cm,
(ft) Description pH (119/9) 25 C)
o - 17 Layered, Fe layers (HCl posi- 7.44 24.2 2279
tive), soft shale layers, sandy
shale
17 . 5- 32.8 Sandy shale; hard carbonaceous 6.21 17.2 2638
shale; clay shale
37.9- 53.1 Sandy and clay shale 9.01 17.2 679
53.1- 70.9 Silty-compact shale 8.84 16.3 825
70.9- 85.2 Sandy shale, hard layered 6.24 15.9 2352
carboniferous shale
85.2-100.2 Nonlayered silty and clay 8.84 21.2 1098
shale
100.2-114.4 Mixed layers of clay, carboni- 8.36 16.2 1354
ferous shale, Fe spots
114.4-131.0 Layered clay, carboniferous 5.73 20.0 3485
shale
131.0-153.1 Coal and compact clay 7.61 16.4 754
153.1-169.7 Silty sandstone, crumbly coal 6.72 20.7 3065
169.7-187.1 Sandstone 8.91 17.5 1078
187.1-200.1 Sandy clay 7.06 22.8 2929
200.1-219.5 Clay, shale, and sandstone 7.15 18.9 4139
219.5-234.2 Sandy shale 8.44 13.6 1609
234.2-254.9 Compact clay, Fe spots 8.78 14.0 1482
254.9-284.8 Silt, coal 8.68 9.0 963
284.8-304.5 Sil tstone, coal 6.69 6.0 3239
22
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Table 7.
Analysis of core sample DH75-106(A).
Depth Pb Conductivity
(ft) Description pH (~g/g) (~mho/cm, 25 C)
5-10 Yellow sand, spots of iron, 8.32 11.7 3881
dark sand in core middle
1 0- 11 Black specks (coal), may be 8.25 11. 3 3518
extraneous from pulling core
out; soft
17 -25 Natural fracture, carboniferous 8.62 7.6 2302
30-31 8.60 6.6 2215
31-32 Carboniferous material-fracture; 8.65 7.9 1514
sedimentary material with
organics
40 Sandstone, soft-moist 8.80 0.0 990
40-41 Carbonaceous material, soft 8.73 12.0 1372
59-60 Hard shale 8.98 7.5 874
72-73 Heavy, dense sandstone, 1 itt1 e 8.99 7.6 833
nonuniformity
78 8.89 5.8 1064
94 Coal, moldy, supporting more 8.41 5.8 1506
life than usual
23
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Table 8. Analysis of core sample DH75-106(B).
Depth Pb Conductivity
(ft) Description pH (l-Ig/g) (l-Imho/cm, 25 C)
o - 15 Silty shale, siltstone; Fe- 7.2 .11. 6 3292
oxide chunks, calcareous spots
15 - 30 Silty, moist shale; Fe-oxidized 7.4 8.0 2170
si1 t
30 - 41 Silty sandstone; Fe-oxidized 8.3 6.2 997
silt
41 - 53.5 Carbonaceous shale, coal; Fe- 7. 1 14.0 1434
oxidized spots
53.5- 71 Shale, some carbonaceous; coal 6.7 10.0 2058
71 - 88 Shale, siltstone 8.8 17.0 720
88 -110.5 Shale, sandstone, gray claystone 9.1 15.5 858
11 0.5-115 Shale with Fe spots and carbona- 7.9 16.5 2058
ceous spots: coal
115 -133.5 Sandstone and shale; carbona- 9.1 14.2 825
ceous spots
133 - 146 Sandstone; carbonaceous streaks, 9.3 9.0 481
mold
146 - 163 Light and gray shale, coal 7.3 14.0 2463
163 -178 Silty and coal shale, gray clay 8.6 11.6 1341
with carbonaceous spot
178 -201.8 Carbonaceous shale 8.9 20.0 743
201.8-233.4 Anderson coal; salt crystals 4.2 13.0 5678
242.6-270.5 Shale with sandstone; carbona- 8.7 11.0 582
ceous streaks
270.5-280 Carbonaceous shale and sandstone 5.3 9.9 2463
24
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Table 9. Analysis of core sample DH76~108.
Depth Pb Conductivity
(ft) Description pH (1l9/9) (llmho/cm, 25 C)
o - 18 Sandy clay; Fe~oxide spots 8.46 11.3 3082
(active HCl reaction), gypsum
precipitate
18 - 20.3 Soft sandstone 8.59 4.9 774
20.3- 43.8 Shale; Fe-oxidized, calcareous 8.09 15.0 2122
and carbonaceous spots
75.8- 79.2 Very carbonaceous shale 3.43 11.5 3422
79.2- 90 Shale and sandstone 8.53 7.2 507
90 - 94.8 Shale in layers 8.07 13.3 1150
94.8-105.5 Clayey sandstone 8.27 2.8 357
105.5-138.6 Shale, carbonaceous shale, 8.72 12.5 595
little coal
25
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Table 10. Analysis of core sample DH75-109.
Depth Pb Conductivi ty
(ft) Description pH (~g/g) (llmho/cm, 25 C)
o - 10 Sandy clay; tan, black carbona- 8.59 10.1 4512
ceous spots, salt spots
10 - 15.5 Sandy clay; black spots, salt 8.59 12.2 3397
spots, orange (Fe) deposits
17 - 22.8 Sandy clay and sand; fine black, 8.54 9.5 2679
shite, and orange granules
22.8- 38.9 Clay; black specks, Fe-oxide 7.52 15.7 3190
deposits, gypsum (separate bag)
38.9- 60.0 Clay, coal, clinker 8.10 8.2 3014
60 -114.6 Siltstone, light shale; carboni- 6.06 8.5 2937
ferous spots, Fe-oxide, gypsum
114.6-122.5 Coal 7.16 3.5 850
122.5- 123. 1 Shale; coal specks 8.59 14.4 451
123. 1- 142.3 Sandy siltstone 9.40 6.9 558
142 . 3- 147 . 5 Shale; carbonaceous streaks 7.83 10.4 1361
147.5-149 Sil tstone 9.51 8.2 472
149 -159 Shale, dark siltstone with coal 8.72 10.4 840
deposits
159 -167.9 Siltstone, clay 9.46 11. 1 524
170 Black shale; calcareous 9.04 13.2 782
streaks
167.9-177.5 Silty siltstone and shale; black 9.65 9.2 458
deposits
177 . 5-200 Silty siltstone and clayey 8.85 9.2 590
siltstone
200 -222.2 Coal, siltstone with coal 7.63 6.8 1129
deposits
222.2-230 Sandy siltstone; black deposits 8.35 6.2 784
230 -243.4 Shale; carbonaceous 8.59 8.8 709
26
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Table 11. Analysis of core sample DH76-111.
Depth Pb Conductivity
(ft) Description pH (fl9/ g) (flmho/cm, 25 C)
o - 0.5 Sandy clay; roots 8.50 57.0 162
0.5- 13.4 Silty sandstone; few Fe-oxidized 9.24 5.5 1383
spots, few calcareous spots
13.4- 30.5 Coal, shale; Fe-oxidized 6.93 15.7 3962
chunks and streaks
30.5- 50 Clayey sandstone 8.89 9.8 1057
50 - 71. 7 Carbonaceous shale 8.96 15.4 587
102 -105 Coal and carbonaceous shale 2.31 20.2 9232
111.5-118 Shale 9.03 12.5 932
126.5-131 .7 Carbonaceous silty shale 8.48 4.8 478
131. 7 -156.5 Shale 8.19 17.8 1001
27
-------
Table 12.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH75-102.
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 10 33 6.24 90 6.59 46 3.00
10- 20 62 6.14 92 5.59 34 2.96
20- 30 96 4.84 93 5.42 38 2.75
N
OJ
30- 40 90 5.06 95 5.50 35 2.90
40- 50 94 4.06 97 2.57 26 2.29
50- 60 94 4.57 73 4.63 16 2.74
60- 70 91 2.97 42 4.54 10 2.35
70- 80 95 2.90 26 4.49 7 2.38
80- 90 92 5.36 97 2.58 18 2.50
90-100 97 2.46 95 3.42 0 2.45
100- 11 0 100 2.78 95 4.10 0 2.37
-------
Table 13.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH75-103.
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosu1 fate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 10 0 6.43 45 5.88 37 3.02
1 0- 20 100 3.14 98 4.10 27 2.95
20- 30 98 4.25 95 3.87 16 2.79
N
~
30- 40 100 2.92 97 2.87 27 2.28
40- 50 93 5.76 73 4.89 9 2.43
50- 60 88 4.98 95 4.30 4 2.40
60- 70 90 5.05 91 4.16 42 3.80
70- 80 100 2.67 4 4.63 0 2.21
80- 90 0 6.19 77 7.62 6 2.54
90-100 89 3.52 25 4.55 4 2.48
100- 11 0 92 3.60 32 4.75 8 2.82
11 0- 120 81 4.38 91 3.41 3 2.30
-------
Table 13. Continued.
Depth S6 S5 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
120-130 90 4.15 87 3.82 12 2.38
130- 140. 95 2.98 87 4.05 7 2.72
w 140-150 94 4.00 51 4.73 8 2.71
o
150- 160 94 5.39 64 4.02 21 2.68
160- 170 20 6.49 54 4.78 6 2.33
170-180 95 3.47 41 4.09 2 2.65
180-188 95 3.12 32 4.28 6 2.75
-------
Table 14.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH75-104.
Depth S6 S5 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 17 96 2.65 93 3.04 23 2.40
17.5- 32.8 92 3.99 65 3.90 11 2.53
37.9- 53. 1 72 6.34 24 4.20 17 2.50
w 53.1-70.9 94 4.01 70 4.67 49 3.28
......
70.9-85.2 95 3.79 63 3.88 34 2.42
85.2-100.2 9 6.35 58 6.08 45 3.07
100.2-114.4 42 6.40 44 6.39 26 2.54
114.4-131.0 97 2.95 89 3.46 10 2.35
131.0-153.1 79 4.05 95 7.15 20 2.65
153. 1- 169. 7 93 6.34 84 4.58 35 2.41
169.7-187.1 93 5.45 96 5.21 60 2.43
187.1-200.1 98 2.45 83 3.80 17 2.30
-------
Table 14. Continued.
Depth 56 55 Fe
eft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
200.1-219.5 92 4.04 62 3.96 33 2.45
219.5-234.2 94 5.02 33 4.22 10 2.50
234.2-254.9 97 5.61 73 4.63 24 2.60
""
N
254.9-284.8 95 2.84 23 4.93 8 2.54
284.8-304.5 90 5.06 95 4.65 56 2.50
-------
Table 15.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH75-106(A).
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
5- 10 89 2.72 10 4.31 13 2.46
1 0- 11 93 5.64 30 4.45 42 2.90
17- 25 0 6.59 0 5.97 38 2.90
w
w
30- 31 0 6.51 0 6.02 32 3.00
31- 32 25 6.85 0 5.80 12 3.45
40 97 6.02 3 6.07 0 2.73
40- 41 97 5.56 12 6.33 5 2.83
59- 60 96 5.74 7 6.12 23 3.28
72- 73 88 5.89 29 7.40 15 3.27
78 95 5.99 6 6.00 3 3.19
94 97 5.74 15 6.56 1 3.11
-------
Table 16.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH75-106(B).
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 15 97 4.95 26 6.68 6 2.75
15- 30 96 5.80 7 6.08 5 2.55
30- 41 93 4.90 3 5.73 0 2.60
t.J
~
41- 53.5 97 5.05 70 6.76 8 2.61
53.5- 71 82 4.70 33 4.91 24 3.26
71- 88 33 6.31 0 6.30 37 3.41
88- 11 O. 5 96 5.42 98 4.70 0 3.25
11 0.5-115 97 6.27 96 5.69 19 2.90
115- 133. 5 54 6.28 14 6.57 47 3.28
133-146 95 5.41 7 5.59 22 3.72
146- 163 66 5.56 49 7.17 35 3.07
163- 178 95 4.00 98 3.73 22 3.05
-------
Table 16. Continued.
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
178-201.8 90 5.80 47 6.30 7 2.51
201 .8-233.4 89 4.51 92 5.82 0 2.34
242.6-270.5 93 5.16 36 5.07 0 2.66
w 270.5-280 84 3.73 69 4.05 0 2.95
U1
-------
Table 17.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH76-108.
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 18 28 5.44 12 5.82 50 3.22
18- 20.3 96 5.61 0 5.87 5 3.34
(...)
en
20.3- 43.8 96 5.49 33 6.05 48 2.60
75.8- 79.2 68 7.07 74 7.03 3 2.21
79.2- 90 47 4.80 45 5.05 26 2.71
90 - 94.8 68 6.82 45 6.97 1 2.38
94.8-105.5 98 3.47 12 6.41 0 2.39
105.5-138.6 96 4.41 55 5.05 0 2.82
-------
Table 18.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH75-109.
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 10 46 6.73 19 6.92 0 2.75
1 0- 1 5 . 5 18 6.71 16 6.07 33 3.05
17- 22.8 44 6.73 9 6.24 69- 3.30
w 22.8- 38.9 95 5.35 19 6.36 0 2.49
"'-J
38.9- 60.0 24 5.87 8 5.99 0 2.52
60-114.6 95 2.62 61 4.30 0 2.37
114.6-122.5 96 5.79 95 7 .10 0 2.60
122.5- 123. 1 59 6.14 45 7.14 0 2.27
123. 1- 142. 3 97 5.76 9 6.10 27 3.35
142.3-147.5 21 7.12 39 6.86 0 2.23
147.5-149 96 5.88 4 6.07 33 3.48
149-159 95 6.45 33 5.25 0 2.65
159-167.9 20 6.32 10 6.19 17 3.34
-------
Table 18. Continued.
Depth 56 S5 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
170 22 6.99 22 6.76 95 7.88
167.9..177. 5 50 6.32 20 6.65 0 2.35
w
(XI
177.5-200 96 5.70 18 5.35 42 3.34
200-222.2 96 3.97 41 5.10 1 2.58
222.2-230 96 4.71 29 6.75 0 2.37
230..243.4 95 4.90 96 6.00 0 2.64
-------
Table 19.
Percent of oxidized thiosulfate and ferrous-iron, and resulting pH from
enrichment cultures of core sample DH76-111.
Depth 56 55 Fe
(ft) Percent pH Percent pH Percent pH
thiosulfate thiosulfate ferrous-iron
oxidized oxidized oxidized
0- 0.5 98 5.43 91 4.07 73 2.33
O. 5- 13. 4 86 5.87 93 5.45 28 3.18
w 13.4- 30.5 90 3.87 31 5.03 18 2.51
~
30.5- 50 92 4.95 91 4.57 39 2.95
50 - 71.7 38 5.92 35 5.55 20 2.66
102 -105 77 7.37 89 6.06 0 2.23
111 . 5- 118 96 5.67 95 5.47 19 2.69
126.5- 131 .7 94 3.91 4 5.81 0 2.49
131.7-156.5 97 4.78 5 5.75 0 2.48
-------
also represented in the 56 enrichment cultures. The 55 medium also produced
representative cultures of these same groups, but the distribution was altered
so that the latter two groups were more prominent. The two extremely acido-
philic groups were not represented in the thiosulfate enrichment cultures,
since none of the resulting pH values were less than pH 2.0. The percentage
of iron oxidized did not exceed 73% when the pH remained below pH 3.0 (Table
19). One sample oxidized 95% of the iron, but had a final pH of 7.88 (Table
18). Purification of the bacteria by repeated streaking was unsuccessful
due to failure of the bacteria to grow adequately on solid media.
Growth of the thiosulfate oxidizing bacteria was ~een on subsequent
transfer of the liquid culture to fresh liquid medium, as well as on transfer
by streak plating of the liquid culture to solidified thiosulfate medium.
Growth was also seen when single, well-isolated colonies from the solid
medium were streaked on fresh solid medium. The subsequent re-streaking of
single, well-isolated colonies for purification resulted in the development
of multiple colony types, which occurred repeatedly with numerous attempts to
purify cultures by streak plate. When single, well-isolated colonies were
transferred to 5 ml of liquid thiosulfate medium in tubes, no growth took
place. The 56 and 55 media were supplemented with yeast extract, but this
did not enhance the development of cultures in liquid medium after growth on
solid medium.
Iron oxidizing bacteria grew in the initial enrichment medium, which
also contained the core sample, but growth on subsequent transfers decreased
until the cultures finally failed to transfer to fresh liquid medium. Yeast
extract, cysteine, glutathione, or IM-MF additives were included in some Fe
media in an attempt to enhance the growth of the cultures, but the cultures
again failed to grow. Purification of the cultures failed due to the inability
of cultures to grow on the solid I5P medium, even after prolonged incubation.
An iron oxidizing bacterial culture isolated from the revegetation site
(1969-11) at Colstrip, Montana was successfully subcultured on 9K or Fe
medium. This isolate also grew in 9K medium supplemented by yeast extract,
cysteine, or glutathione although these growth additives were not required.
No growth occurred when IM-MF additives were included in the medium. The
culture oxidized 99% of the ferrous iron and produced a final pH of 1.84.
Algal Growth
Algal growth curves. Figure 3 shows the algal growth curves of SeZena-
strum aapPiaornutum Printz determined prior to the algal bioassays of leachates.
Parameters of algal growth were: in vivo chlorophyll a fluorescence, extracted
chlorophyll a fluorescence~ extracted phaeopigment a absorbance, turbidity,
cell count and ash free welght. In vivo fluorescence indicated a maximum
standing crop on day 5. Extracted chlorophyll a fluorescence and absorbance
bot~ :eache~ maximum v~lues on day 4. Ce~l number and turbidity curves showed
a slmllar rlse, but falled to reach a maXlmum. Extracted phaeopigment a
absorbance and ash free weight did not follow the growth of the algal culture.
40
-------
..
t: i
z
~ I
III ~
u
i z
III ~
.... II! ..
..
... III .! ..
iiI i 5 . i
u .. ~ ~
.... ~ ...
~ . ... i
.! ~
III :II:
II: .. i! ..
III :II: !c 0 .
.. " ~ I
2 ;;; ...
~ W
z . t
101' ~ 10' u
100 100
10'
102
10' .,.. 10 I
2
3
DAY
4
&
6
10""1 10""1
IO-t at
Algal growth curves of SeZenastrum aapriaoPnutum Printz, comparing six
methods of biomass monitoring. [In vivo chlorophyll a fluorescence
(~), extracted chlorophyll a fluorescence (0---0), extracted chloro-
phyll a absorbance (A665, ~), extracted phaeopigment a absorbance
(A665,<:r-"~), turbidity (A750, 0---0), cell count (0----0), and ash
free weight (o---o}.J
Fig. 3.
41
-------
Algal growth studies. Algal growth studies to determine the influence of
pH, glassware acid washing, inoculum size, inoculum age, and inoculum washing
are illustrated in Figure 4. In the pH study, a'six-day-old inoculum was used.
All acid pH values (3.6, 4.2, 4.6, 5.0, and 5.4) completely inhibited the alga.
Using a six-day-old culture as inoculum, the standing crop obtained was not
increased by doubling the inoculum size. Two- or four-day-old inoculum gave
a greater maximum standing crop than a six-day-old inoculum. Two- and four-
day-old inocula produced approximately equal growth curves and maximum
standing crop. The EPA recommended inoculum washing (U.S. EPA 1971), had no
effect on the maximum standing crop.
Algal bioassays control curves. Figure 5 shows the irregularity in the
algal controls when a six-day-old culture was used as the inoculum. Two-day-
old cultures consistently produced and increased biomass.
leaching Studies
Thirty of the 110 core samples were used in the leaching studies. These
samples represented a wide range of pH value, lead content, and conductivity.
Tables 20 through 27 show the results of the chemical analyses of the core
samples and leachates and the fluorometric analysis of the leachate algal bio-
assays. The pH values of the core samples selected for leaching studies ranged
from 2.31 to 9.3 (Tables 27 and 24, respectively), with most pH values between
5.3 and 8.5. All lead values in leachates represent the concentration in ~g/g
of core leached, not the actual concentration in the leach solution. Most core
samples had lead concentrations of 9.0 to 20.7 ~g/g and the maximum was 57.0
pg/g (Tables 23 and 27). Conductivity of the samples ranged from 162 ~mhos/cm
to 9232 ~mhos/cm (Table 27), with most samples between 1372 ~mhos and 3962
pmhos/cm. The core samples selected encompassed the entire range of each of
the chemical parameters analysed, viz. pH, lead, and conductivity.
Table 20 shows the pH values and lead content of the leachates from core
sample DH75-102. The leach medium for this core contained glucose. Two
flasks had the autotrophic inoculum and two had the soil inoculum. The recorded
values are an average of the two respective results. The shaking autotrophic
control of the 40-50 ft. sample caused a decrease in pH below 4.0, which was
arbitrarily designated as the boundary between low and high pH values. Some
of the other controls caused a decrease in pH, but none of these resulted in
a low pH. The lead contents of the leachates were roughly similar and were
not proportional to the Pb content of the core samples. The highest Pb was
in the leachate of the shaking soil control of the 50-60 ft. sample, and was
4.65 pg/g. The.average amount of Pb in the leachates was 2.60 ~g/g, with the
stationary leachates averaging 2.07 pg/g and the shaking 1eachates averaging
3.13 ~g/g. The Pb values grouped according to inoculum were: uninoculated
2.16 ~g/g, autotrophic inoculum 2;52 ~g/g, and soil inoculum 3.12 ~g/g.
Stationary and shaking conditions were: stationary uninoculated 1.86 ~g/g,
stationary autotrophic inoculum 1.92 pg/g, and stationary soil inoculum 2.40
pg/g, and shaken uninocu1ated 2.46 ~g/g, shaken autotrophic inoculum 3.09 ~g/g,
and shaken soil inoculum 3.84 ~g/g.
Table 21 shows the results of the leaching of DH75-103. The pH values
and Pb content of the leachates varied relatively little, with no 1eachates
below pH 4.0 and with Pb concentrations which were proportional to the Pb
42
-------
103
U)
~
z
=>
UJ
U
Z
UJ
U
U)
UJ 102
a:
o
=>
...J
lL.
UJ
>
ti d
...J
UJ
a:
100
...0- -- -0- -- - {}- - - - {]
,~
l::r--- -6
,,t:r~'~~
,
/' "
. ..lJ.'
.~
,.
2
3
4
S
DAY
6
7
8
9
Algal growth studies of Selenastrum capricornutum Printz, determining
the influence of five factors on algal growth, as measured by in vivo
fluorescence method. [Two-day-old inoculum (0): washed inoculum (0---0)
and unwashed inoculum (0----0); four-day-old inoculum (0): washed
inoculum (0---0) and urowashed inoculum (0---0); and six-day-old inocu-
lum (~): IX inoculum (~---~) and 2X inoculum (~) in chromic-
HC1-HN03 acid washed glassware. AAP medium at pH 3.6, 4.2, 4.6, 5.0,
and 5.4 (~...~) and AAP medium at pH 7.6 (~--.~).]
Fi g. 4.
43
-------
103
en
"' ~
z
~
LIJ
(.)
Z
LIJ
(.)
en
LIJ
~ 102
~
..J
I&..
LIJ
>
~
..J
LIJ
II:
101
- D- - [Y"'"'-O........'
0- - [Y"'" -- . "iJ
p--.if '
r/'
. cY",o
-g-::------6
"r- -
l::r-'''/s' ,
,/
a"'"
, ,0
,0"'-
,
,
,
,
'.... ,,"
... "
'cf
2
3
4
5
6
DAY
7
8
9
10
II
12
Algal control growth curves of Selenastrum aapriaornutum Printz, used
with different leachate bioassays as determined by in vivo fluorescence
measurements. [Six-day-old inoculum (0 and ~): 12-5-76 (o---o),
1-8-77 (tr-----l1), 2-16-77 (o---o), 3-25-77 (~---~), 4-29-77 (o---o);
and two-day-old inoculum (o): 1-21-78 (0---0), 1-31-78 (o---o), and
2-11-78 (0-- -o).J
Fig. 5.
44
-------
Table 20. Core and leachate analyses and algal bioassays, core sample DH75-102.
GS VI-VII Medium Algal BioassaYSa
Depth Sample Inoculum With or Wi thout Static Leachate Shaking Leachate Static Leac~ate Shaking Leaghate
(ft) pH Pb (1Jg!g) Description 0.1% Glucose pH Pb (1Jg!g) pH Pb (1Jg!g) Percent Percent
40- 50 5.47 2.70 Uninocu1ated With 5.80 1.50 4.83 2.40 498 -86
Autotrophic 5.72 1. 50 2.90 1.95 818 -98
inocu1umc
Soil inoculum 5.06 2.10 4.43 2.70 586 308
50- 60 8.46 14.40 Uninocu1ated With 8.35 1.50 8.02 2.40 39 418
Autotrophi c 8.26 2.10 7.89 3.45 -12 8
~ inoculum
U1
Soil inocu1 um 6.97 2.40 5.91 4.65 -14 22
80- 90 7.29 2.80 Uninoculated With 8.01 2.40 6.41 1.80 715 535
Autotrophic 7.67 1.80 5.59 3.30 0 249
inoculum
Soil inoculum 6.09 2.40 5.71 4.20 -4 43
90-100 7.41 4.80 Uninoculated With 8.03 2.10 7.83 2.70 0 51
Autotrophic 8.06 2.10 7.35 3.75 373 469
inoculum
Soil inoculum 6.15 2.25 5.77 4.05 16 35
-------
Table 20. Continued.
GS VI-VII Medium Algal Bioassays
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leachate Shaking Leachate
(ft) pH Pb (\Jg/g) Description 0.1% Glucose pH Pb (\Jg/g) pH Pb (\Jg/g) Percent Percent
1 00- 11 0 7.48 12.40 Uninocu1ated With 7.92 1.80 6.24 3.00 49 -27
Autotrophic 7.66 2.25 5.70 3.00 198 4
inoculum
~ Soil inoculum 5.66 2.85 6.37 3.60 10 20
0'1
aInhibition is indicated by a negative value, whereas stimulation is indicated by a
bInocu1um taken from a six-day-01d culture.
CAutotrophic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
positive value.
-------
Table 21. Core and leachate analyses and algal bioassays. core sample DH75-103.
GS VI-VII Medium Algal BioassaYSa
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leachate Shaking Leachate
(ft) pH Pb (~g/g) Description 0.1% Glucose pH Pb (~g/g) pH Pb (~g/g) Percentb Percentb
10- 20 8.32 23.8 Uninoculated Wi thout 6.19 2.10 6.91 1.80 -9 6
Autotrophic 6.71 1.50 7.16 1.80 -47 31
inoculum<:
Soil inoculum 6.17 2.10 7.48 1.80 -35 28
Uninoculated With 8.59 2.70 6.06 2.40 21 79
~ Autotrophic 8.65 2.40 6.92 2.10 162 44
....... inoculum
SoH inoculum 8.21 2.70 7.24 2.10 -30 12
1 00- 11 0 8.49 49.1 Uninoculated Without 5.65 3.00 7.83 2.10 -30 618
Autotrophic 7.25 3.30 7.96 2.70 -17 188
inoculum
Soil inoculum 6.37 3. ('~ 7.96 3.60 -55 83
Uninoculated With 8.18 3.00 5.92 1.50 3 169
Autotrophic 8.17 3.00 6.18 1.80 -10 102
inoculum
Soil inoculum 7.45 3.60 6.89 2.70 -62 79
-------
Table 21. Continued.
GS VI-VII Medium Algal BioassaYSa
Depth Sample Inoculum Wi th or Wi thout Static Leachate Shaking Leachate Static Leac~ate Shaking Leachate
(ft) pH Pb (\.Ig/g) Description 0.1% Glucose pH Pb (\.Ig/g) pH Pb (\.Ig/ g) Percent percentb
120-130 6.23 24.3 Uninoculated Wi thout 6.88 3.00 7.36 3.00 -25 -10
Autotrophic 6.85 3.90 6.67 3.60 -10 73
inoculum
Soil inoculum 5.23 4.05 7.06 3.90 -37 60
Uninoculated With 6.83 3.90 6.97 2.40 6 51
~ Autotrophic 6.54 4.05 6.68 3.90 -0.3 62
00
inoculum
Soi 1 i nocul urn 6.70 4.50 6.59 3.90 11 27
aInhibition is indicated by a negative value, whereas stimulation is indicated by a positive value.
bInoculum taken from a six-day-old culture.
CAutotrophic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
-------
Table 22. Core and leachate analyses and algal bioassays. core sample DH75-104.
GS VI-VII Medium A1fia1 BioassaYSa
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leac ate Shaking Leachate
(ft) pH Pb (~g/g) De$cription 0.1% Glucose pH Pb (~g/g) pH Pb (~g/g) Percentb Percenf.C
0- 17 7.44 24.2 Uninoculated Without 7.63 1.50 6.62 1.50 -9 144
Autotrophic 7.71 '1.50 6.15 1.80 25 6
inoculumd
Soil inoculum 6.57 1.80 7.05 2.10 -27 15
Uninoculated With 7.80 2.10 5.95 4.65 -67 83
Autotrophic 7.57 2.40 6.87 2.40 -10 31
~ inoculum
1.0
Soil inoculum 6.74 2.70 7.44 2.10 -30 -7
114.4-131.0 5.73 20.0 Uninoculated Without 3.22 3.90 3.25 3.30 -95 -99
Autotrophic 3.17 2.40 2.54 4.80 -95 -99
inoculum
Soil inoculum 4.01 2.10 7.04 4.20 -96 -34
Uninoculated With 3.31 2.4'0 2.89 5.10 -95 -99
Autotrophic 3.31 2.10 2.56 4.80 -96 -99
inoculum
Soil inocul um 4.92 1.80 7.17 3.66 -93 -58
-------
Table 22. Continued.
pH
Sample
Pb (\.Ig/g)
Inoculum
Description
GS VI-VII Medium
With or Without
0.1% Glucose
Static Leachate
pH Pb (\.Ig/g)
Shaking Leachate
pH Pb (\.Ig/g)
Algal BioassaYSa
Static Leachate Shaking Leachate
Percentb PercentC
Depth
(ft)
153.1-169.7 6.72 20.7 Uninoculated Without 7.35 1.20 7.41 2.70 58 -78
Autotrophic 7.36 1.80 7.49 3.30 46 -96
inoculum
Soil inoculum 6.13 2.40 7.16 2.40 8 -77
Uninoculated With 8.06 1.80 7.70 3.00 139 -69
Autotrophic 8.17 2.10 7.60 3.60 50 -77
inoculum
Soil inoculum 7.33 2.40 7.46 3.30 54 -70
U'1 200.1-219.5 7.15 18.9 Uninoculated Without 6.97 0.60 7.07 1. 50 -9 -82
o
Autotrophic 7.02 0.90 7.35 2.10 31 -83
inoculum
Soil inoculum 5.33 0.90 6.85 2.70 45 57
Uninoculated With 8.19 1. 50 7.67 2.10 61 -83
Autotrophic 8.05 2.10 7.22 1.50 46 -79
inoculum
Soil inoculum 7.90 2.10 6.87 2.70 20 45
-- - - ------- ---- -----
alnhibition is indicated by a negative value, whereas stimulation is indicated by a positive value.
bInoculum taken from a six-day-old culture.
cInoculum taken from a two-day-old culture.
dAutotroPhic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
-------
Table 23. Core and leachate analyses and algal bioassays, core sample DH75-106(A).
GS VI-VII Medium Algal BioassaYSa
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leaccate Shaking Leachate
(ft) pH Pb (Ilg/g) Description 0.1% Glucose pH Pb (Ilg/g) pH Pb (Ilg/g) Percent PercentC
5- 10 8.32 11.7 Uninoculated Without 7.78 0.90 7.76 2.70 85 -77
Autotrophic 7.75 1.50 7.62 3.00 -57 -85
inoculumd
So11 inoculum 5.51 2.10 6.85 3.90 335 48
Uninoculated With 8.16 1.80 8.28 2.70 68 -83
U1 Autotrophi c 8.04 2.10 8.11 2.70 205 -83
.- inoculum
So11 i nocul um 6.96 2.40 7.07 3.60 95 51
10- 11 8.25 11.3 Un i noc'u 1 a ted Without 8.08 0.90 8.09 0.60 -10 88
Autotrophic 8.09 0.90 8.00 0.60 8 68
inoculum
Soil inoculum 6.04 1.50 7.06 1.50 -63 86
Uninoculated With 8.58 1.20 8.40 3.60 650 -81
Autotrophic 8.52 1.80 8.64 3.60 -90 0
inoculum
Soil inoculum 7.89 1.50 7.39 1.50 30 54
-------
Table 23. Continued.
GS VI-VII Medium Algal Bioassays
Depth Sample Inoculum Wi th or Wi thout Static Leachate Shaking Leachate Static Leachate Shakinq Leachate
(ft) pH Pb (~g/g) Description o.a; Glucose pH Pb (~g/g) pH Pb (~g/g) Percent Percent
40 B.BO 0.00 Uninocul ated Without 8.01 0.60 8.14 2.10 43 297
Autotrophic 7.99 0.60 8.22 0.90 2 176
inoculum
Soil i nocul um 5.42 0.90 7.07 1.50 18 -17
Uninoculated With 8.39 1.20 8.17 1.20 250 -1
Autotrophi c 8.33 0.90 8.35 3.00 48 208
inoculum
Soil inocul urn 8.16 1.20 5.82 4.20 70 -1
U'1 40- 41 8.73 12.0 Uninoculated Without 7.98 0.90 8.11 1.20 79 -62
N
Autotrophic 8.03 1.80 6.92 1.20 44 146
inoculum
Soil inoculum 5.30 1.80 7.99 1.50 53 114
Uninoculated With 8.20 1. 50 8.39 0.90 33 -91
Autotrophic 8.13 1.80 8.41 0.90 7 241
inoculll11
Soil inoculum 7.28 1.80 7.22 0.90 -67 -86
alnhibition is indicated by a negative value, whereas stimulation is indicated by a positive value.
bInoculum taken from a six-day-old culture.
clnoculum taken from a two-day-old culture.
dAutotroPhic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
-------
Table 24. Core and leachate analyses and algal bioassays, core sample DH75-106(B).
GS VI-VII Medium A1ga1.Bioassaysa
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leac~ate Shakin9 Leachate
(ft) pH Pb (~g/ 9) Description 0.1% Glucose pH Pb (~9/9) pH Pb (~9/9) Percent percentb
71-88 8.80 17.0 Uninocu1ated Without 7.10 3.60 6.60 1.80 16 -99
Autotrophic 8.47 4.80 7.21 3.00 14 68
i noculLlnc
Soil inocul urn 5.85 5.40 7.98 2.70 14 17
Uninocu1ated With 8.80 4.50 8.26 ~.40 32 -9
(J"I Autotrophic 8.61 4.20 5.75 4.50 29 51
w inoculum
Soil inoculum 7.61 3.00 8.34 2.40 29 48
133-146 9.30 9.00 Uninpcu1ated Without 8.43 3.00 8.41 0.00 75 -4
Autotrophic 8.32 2.70 7.65 0.00 19 38
inoculum
Soil inoculum 5.92 4.20 6.91 0.00 83 -62
Uninocu1ated With 8.69 3.00 8.06 0.00 29 -83
Autotrophic 8.61 2.70 7.82 3.00 42 28
inoculum
Soil inoculum 7.47 3.00 8.21 3.00 53 -98
-------
Table 24. Continued.
GS VI-VII Medium Algal Bioassays
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leachate Shaking Leachate
( ft) pH Pb (\Jg/g) Description 0.1% Glucose pH Pb (\Jg/ 9 ) pH Pb (\Jg/g) Percent Percent
178-201.8 8.90 20.0 Uninoculated Without 6.04 3.00 6.22 3.60 46 -49
Autotrophic 5.87 1.50 6.14 3.60 -1 -35
inoculum
Soil inoculum 5.26 3.60 5.75 3.30 66 -84
Uninoculated With 8.07 1.50 7.72 2.70 66 -1
Autotrophic 7.62 2.10 3.53 5.40 -7 -62
inoculum
U1
~ Soil inocul um 6.43 2.70 7.49 2.70 55 42
201.8-233.4 4.20 13.0 Uninoculated Without 2.83 3.00 3.18 4.50 -99 -100
Autotrophic 2.35 0.00 2.31 6.00 -99 -100
inocul urn
Soil inoculum 2.31 2.10 4.41 3.00 -99 -98
Uninoculated With 2.77 6.60 3.91 3.00 -100 -59
Autotrophic 2.36 5.10 2.75 4.50 -99 -98
inoculum
Soil inoculum 2.45 6.00 3.94 3.00 -99 -14
-------
Table 24.
Continued.
U'1
U'1
GS VI-VII Medium Algal Bioassays
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leachate Shaking Leachate
( ft) pH Pb (~g/g) Description 0.1% Glucose pH Pb (~g/g) pH Pb (~g/g) Percent Percent
J
270.5-280 5.30 9.90 Uninocu1ated Without 7.08 1.20 7.19 2.70 6 4
Autotrophic 7.20 2.70 6.37 3.00 -12 -64
inoculum
Soil inocu1 urn 5.21 2.40 4.92 2.70 6 -98
Uninocu1ated With 6.49 3.00 4.52 5.10 6 -86
Autotrophic 5.97 2.70 4.92 4.50 -98
inoculum
Soil inoculum 5.05 3.00 6.54 3.00 12 3
alnhibition is indicated by a negative value, whereas stimulation is indicated by a
blnoculum taken from a two-day-01d culture.
CAutotrophic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
positive value.
-------
Tab1e 25; . Core and 1eachate analyses and algal bioassays, core sample DH76-108.
GS VI-VII Medium A1ga1 BioassaYSa
Depth Sample Inocu1um With or Without Static Leachate ~akingp~e(chr) Static Leac~ate Shaking Leachate
(ft) pH Pb (~g/g) Description 0.11 G1ucose pH Pb (~g/g) pH ~gg P~rcent Percentb
0- 18 8.46 11.3 Uninoculated Wi thout 8.56 4.20 6.96 3.90 -54 150
Autotrophic 8.48 2.70 7.71 2.40 -53 140
inoculum<:
Soil inoculum 5.70 2.70 7.54 2.10 -74 241
Uninoculated With 5.78 2.10 7.51 3.00 -76 195
(J'1
0"1 Autotrophic 6.65 3.90 7.65 1.80 38 411
inoculum
Soil inoculum 6.22 5.10 7.52 2.10 -32 64
20.3-43.8 8.09 15.0 Uninoculated Without 7.67 2.70 7.86 1.20 70 61
Autotrophic 7.84 2.10 5.98 2.70 113 39
inoculum
Soil inoculum 5.42 4.50 6.74 1.80 -59 114
Uninoculated With 8.50 2.10 8.55 1.20 43 57
Autotrophic 8.18 4.50 8.32 1.50 -16 82
inoculum
Soil inoculum 5.67 3.90 7.17 2.70 -42 286
-------
Table 25.
Continued.
U'1
""-J
GS VI-VII Medium Algal Bioassays
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leachate Shaking Leachate
( ft) pH Pb (\.Ig/g) Description 0.1% Glucose pH Pb (\.Ig/g) pH Pb (\.Ig/g) Percent Percent
75.8-79.2 3.43 11.5 Uninoculated Without 2.98 5.40 2.88 7.20 -96 -77
Autotrophic 2.83 5.10 2.29 4.50 -94 -77
inoculum
Soil inoculum 4.71 3.00 4.41 2.10 -77 159
Uninoculated With 3.02 2.10 2.76 1.50 -87 -80
Autotrophic 3.06 2.70 2.24 2.40 -91 -86
inoculum
Soil inocul urn 6.03 1.20 4.40 1.20 -46 -45
alnhibition is indicated by a negative value, whereas stimulation is indicated by a
blnoculum taken from a six-day-old culture.
CAutotrophic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
positive value.
-------
Table 26. Core and leachate analyses and algal bioassays, core sample DH75-109.
GS VI-VII Medium Algal BioassaYSa
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leac~ate Shaking Leachate
(ft) pH Pb (\lg/g) Description 0.1% Glucose pH Pb (\lg/g) pH Pb (\lg/gf Percent PercentC
22.8-38.9 7.52 15.7 Uninoculated Without 5.91 0.90 7.63 2.10 -86 -53
Autotrophic
inoculumd 5.97 1.20 7.74 2.10 -90 2
5011 inoculum 6.13 2.10 4.86 3.30 -53. -6
Uninoculated With 8.30 1.50 8.08 3.60 -77 1413
Autotrophic 8.24 2.40 8.22 3.00 -55 15
i noculllll
Soil inoculum 6.43 2.40 6.93 2.40 -58 1746
U1 60- 114 . 6 NDe
(X) 6.06 8.50 Uninoculated Wi thout 7.36 1.50 8.17 0.30 2
Autotrophic 5.93 2.40 7.88 0.90 -3 102
inoculum
Soil inoculum 6.26 2.70 4.48 0.30 6 291
Uninoculated With 7.93 2.10 7.49 0.90 -46 9
Autotrophic 7.93 2.70 6.65 0.30 -50 -7
inoculum
Soil inoculum 6.74 2.40 4.61 1.20 0.4 ND
---.. --------.. ~---~-
alnhibition is indicated by a negative value, whereas stimulation is indicated by a positive value.
b
Inoculum taken from a two-day-old culture.
clnoculum taken from a six-day-old culture.
dAutotrophic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
eNot determined.
-------
Table 27. Core and leachate analyses and algal bioassays, core sample DH76-111.
GS VI-VII Medium Algal Bioassaysa
Depth Sample Inoculum With or Without Static Leachate Shaking Leachate Static Leac~ate Shaking Leachate
(ft) pH Pb (~gl g) Description 0.1% Glucose pH Pb (~g/g) pH Pb (~g/g) Percent PercentC
0-0.5 8.50 57.0 Uninoculated Without 6.20 1.20 6.13 1.80 -30 136
Autotrophic 6.19 2.10 6.24 3.60 -10 255
inoculuind
Soil inoculum 5.19 0.90 6.42 3.00 -36 84
Uninoculated With 8.47 2.70 6.14 2.10 58 132
Autotrophic 8.47 0.90 7.08 2.70 22 155
i noculll11
Soil inoculum 6.00 0.90 6.77 3.30 20 150
U1 13.4-30.5 6.93 15.70 Uninoculated Without 7.64 2.10 7.77 3.30 -29 355
\0
Autotrophi c 7.55 0.90 7.80 2.40 -88 150
inoculum
Soil i nocul um 5.36 1.80 7.31 2.40 -46 86
Uninoculated With 7.51 1.80 7.39 2.10 -65 118
Autotrophic 7.44 3.30 7.43 1.80 -96 655
inoculum
Soil i nocull111 5.53 3.60 6.64 3.90 -50 655
-------
Table 27. Continued.
GS VI-VII Medium Algal Bioassays
Depth Sample Inoculum Wi th or Wi thout Static Leachate Shaking Leachate Static Leachate Shaking Leachate
(ft) pH Pb (\1g/g) Description 0.1% Glucose pH Pb (\1g/g) pH Pb (\1g/g) Percent Percent
102-105 2.31 20.20 Uninoculated Wi thout 2.27 2.10 2.09 8.40 -100 -86
Autotrophic 2.26 2.10 1.62 10.5 -100 -98
inocul urn
Soil inoculum 2.71 1.80 1.85 9.00 -100 -93
Uninoculated With 2.33 3.00 2.14 5.70 -100 -100
Autotrophic 2.18 2.70 1.61 9.30 -100 -98
i nocul urn
0'1 Soil inoculum 2.96 3.60 1. 95 9.30 -100 -89
a
131.7-156.5 8.19 17.80 Uninoculated Without 7.37 3.00 7.16 4.50 -10 -4
Autotrophic 7.05 1. 20 6.03 2.40 -7 9
inoculum
Soil inoculum 4.87 2.40 3.94 6.00 -51 25
Uninocu1ated With 6.79 2.70 5.48 3.90 -7 3
Autotrophic 6.35 3.90 4.67 4.50 -10, -13
inoculum
Soil i nocul urn 5.38 0.90 5.71 3.60 -44 -7
aInhibition is indicated by a negative value, whereas stimulation is indicated by a positive value.
b
Inoculum taken from a two-day-01d culture.
cInocu1um taken from a six-day-01d culture.
dAutotroPhic inocula are a mixture of sulfur and ferrous iron oxidizing bacteria.
-------
content of the core. The average amount of Pb in the leachates was 2.86
~g/g, with stationary leachates averaging 3.10 ~g/g and shaking leachates
2.62 ~g/g. The three different leachates, averaging stationary and shaking
conditions together, produced average Pb concentrations in the uninoculated
flasks of 2.58 ~g/g, in the autotrophic inoculum flasks of 2.84 ~g/g, and
in the soil inoculum flasks of 3.26 ~g/g. The addition of glucose produced
little changes in Pb values. Specifically. the flasks without glucose were
2.79 ~g Pb/g and the flasks with glucose were 2.93 ~g Pb/g.
The leaching of DH75-104 resulted in an average Pb concentration of 2.46
~g/g with some leachates going below pH 4.0 (Table 22). Several of the flasks
in the 114.4-131.0 ft. sample produced acid, with pH as low as 2.54. The soil
inoculum flasks for this sample depth did not produce these low pH values.
However, the shaken flasks whether uninoculated or with an autotrophic inoculum
produced generally low pH values. The shaken controls also showed higher Pb
concentrations than did the stationary controls, 3.96 ~g/g and 1.94 ~g/g,
respectively. When the Pb values for shaken and stationary flasks were com-
bined and arranged by inoculum, the results were: uninoculated 2.43 ~g/g, the
autotrophic inoculum 2.48 ~g/g, and the soil inoculum 2.47 ~g/g. The flasks
with glucose added to the medium had a composite Pb value of 2.69 ~g/q, where
the flasks without glucose had 2.27 ~g/g.
In Table 23 leachates from DH75-106(A) show similar pH values to the
pH values found in other leachates, but lower Pb concentrations. The average
Pb content was 1.74 ~g/g. The stationary and shaken flasks had average Pb con-
centrations of 1.31 ~g/g and 2.06 ~g/g, respectively. The average Pb concen-
trations from the flasks with the thr~e different inocula were 1.51 ~g/g for
the uninoculated flasks, 1.71 ~g/g for the autotrophic inoculum flasks, and
1.99 ~g/g for the soil inoculum flasks. The medium without glucose resulted
in an average Pb concentration of 1.47 ~g/g, while the medium with glucose
resulted in 2.00 ~g/g.
In Table 24 core samples from DH75-106(B) are shown to produce some
leachates below pH 4.0 and some with Pb concentrations as high as 6.0-6.6 ~g/g.
Leachates with pH values below pH 4.0 were observed in several of the 201.8-
233.4 ft. sample flasks and in the 178-201.8 ft. sample with shaking incuba-
tion of the autotrophic inoculum with glucose. The average Pb concentration
in the leachates was 3.09 ~g/g. The stationary and shaking flasks had Pb con-
centrations of 3.21 ~g/g and 2.97 ~g/g, respectively. The inoculum flasks had
average Pb concentrations of 2.91 ~g/g for the uninoculated flasks, 3.30 ~g/g
for the autotrophic inoculum flasks, and 3.06 ~g/g for the soil inoculum flasks.
The average Pb concentration for the different media was 2.77 ~g/g when glucose
was omitted and 3.41 ~g/g when glucose was included.
Core sample DH76-108 in Table 25 had one sample depth, 75.8-79.2 ft., with
leachate pH values below 4.0. The leachate from this sample depth, uninoc-
ulated, without glucose, and incubated on the shaker, had a Pb concentration
of 7.20 ~g/g. The average Pb concentrations in the leachates were 3.35 ~g/g
and 2.52 ~g/g, respectively. Inoculum results were 3.05 pg/g for the uninocu-
lated, 3.03 ~g/g for the autotrophic inoculum, and 2.70 ~g/g for the soil
inoculum. Values of 3.41 ~g/g and 2.50 ~g/g were found for media without
and with glucose, respectively.
61
-------
The leaching of DH75-109 in Table 26 resulted in an average Pb concen-
tration of 1.87 ~g/g with no pH below 4.0. Stationary and shaking flasks
averaged Pb concentrations at 2.03 ~g/g and 1.70'~g/g, respectively. The
different inocula had Pb concentrations of 1.62 ~g/g for the uninoculated
flasks, 1.88 ~g/g for the autotrophic inoculum flasks, and 2.10 ~g/g for the
soil inoculum flasks. The different media, without glucose and with glucose,
resulted in 1.65 ~g/g and 2.08 ~g/g, respectively.
Most sample depths of core DH76-111 (Table 27) resulted in approximately
equal pH values. However, sample depth 102-105 ft had pH values below pH 4.0
and Pb concentrations as high as 10.50 ~g/g. The shaking autotrophic inoculum
flasks of this sample depth had Pb concentrations of 10.5 ~g/g and 9.30 ~g/g
for the without and with glucose media flasks, respectively. The average Pb
concentration found in the 1eachates from this core was 3.36 ~g/g. Stationary
and shaking flasks had average Pb concentrations of 2.15 ~g/g and 4.57 ~g/g,
respectively. The flasks had average Pb concentrations of 3.16 ~g/g for the
uninocu1ated, 2.40 ~g/g for the autotrophic inoculum, and 3.53 ~g/g for the
soil inoculum. The flasks with the different media were very similar in average
Pb content with 3.29 ~g/g in the without glucose flasks and 3.43 ~g/g in the
with glucose flasks.
The similarity between the leaching of the different core samples is
shown by Tables 20-27. Many samples had a pH drop to below 4.0, while most
samples did not alter the pH of the leaching solution appreciably. The pH
of the 1eachates ranged from pH 1.61 to 8.8 (Tables 27 and 24, respec-
tively). Pb concentrations of the 1eachates ranged from 0.0 ~g/g to 10.50
~g/g (Tables 24 and 27, respectively), with the overall average Pb concentra-
tion of 2.66 ~g/g of core.
Algal Bioassays of leachates
The results of the algal bioassays for toxicity of leachates from core
samples are included in Tables 20-27. The bioassays are reported as percent
inhibition, shown by a negative value, or percent stimulation, shown by a
positive value. The inoculum of the bioassay experiments was taken from a
six-day-01d culture or a two-day-01d culture; this is noted on each table.
The algal bioassays of the leachates from core DH75-102, using a six-day-
old culture as inoculum, are shown in Table 20. Some 1eachates were toxic,
but most leachates showed either no effect or a stimulation of algal growth.
Stimulation was as high as 818% for the stationary, soil inoculum flask of
sample depth 40-50 ft and 715% for the stationary, uninocu1ated flask and
535% for the shaking, uninocu1ated flask of sample depth 80-90 ft. The
inhibition of 86% and 98% by the 40-50 ft sample was produced by 1eachates
from the shaken uninocu1ated and shaken autotrophic inoculum flasks, with pH
values of 4.83 and 2.90, respectively, and Pb contents of 2.40 P9/9 and 1.95
~g/g, respectively. The other 1eachates showing inhibition had pH values above
pH 6.09 and Pb values below 2.40 pg/g.
Table 21 shows the bioassays from core DH75-103, using a six-day-01d
inoculum. Most 1eachates stimulated algal growth with values up to 618% for
the shaken, uninocu1ated, no glucose flask of sample depth 100-110 ft and 162%
62
-------
for the stationary, autotrophic, with glucose flask of sample depth 10-20 ft.
Inhibition was as high as 55% with the stationary, soil inoculum, no glucose
flask of sample depth of 100-110 ft. The pH values of the leachates ranged
from 5.23 to 8.21 and the Pb concentration ranged from 1.50 ~g/g to 4.05 ~g/g.
Table 22 shows the results of the algal bioassays of core samples from
DH75-104, using a six-day-old culture as inoculum for the stationary leachate
bioassays and a two-day-old culture as the inoculum for the shaking leachate
bioassays. Stimulation was 139% for the stationary, uninoculated, with glu-
cose flask of sample depth 153.1-169.7 ft and 144% for the shaking, uninocu-
lated, no glucose of sample depth 0-17 ft. However, most of these leachates
were toxic, with many having inhibition values of 90%. The leachates of
sample depth 114.4-131.0 ft, which had greater than 90% inhibition, had pH
values below 4.92 and Pb values ranging from 1.80 ~g/g to 5.10 ~g/g. The other
inhibitory leachates had pH values ranging from 6.57 to 7.80 and Pb values
from 0.60 ~g/g to 3.60 ~g/g. The leachate of the stationary. uninoculated,
without glucose flask from sample depth 200.1-219.5 ft had a pH value of
6.97 and a Pb content of 0.60 pg/g and produced 9% inhibition. The leachate
of the shaking, autotrophic inoculum, without glucose flask from sample depth
153.1-169.7 ft had a 98% inhibitory effect.
Table 23 gives algal bioassays of leachates from core DH75-106(A).
Stationary leachates used a six-day-old inoculum and the shaking leachates a
two-day-old inoculum. Stimulation was 650% for the stationary, uninoculated,
with glucose flask of sample depth 10-11 ft, and 335% for the stationary,
soil inoculum, without glucose flask of sample depth 5-10 ft, and 297% for
the shaking, uninoculated, without glucose flask of sample depth 40 ft. The
pH values and Pb concentrations for these flasks showing stimulation were pH
8.58 and 1.20 ~g/g, pH 5.51 and 2.0 ~g/g, and pH 8.44 and 2.10 ~g/g, respec-
tively. The inhibition by these core leachates ranged from 1% to 91%. The
leachates showing inhibition had pH values ranging from 5.82 to 8.52, and
Pb content ranging from 0.90 ~g/g to 4.20 ~g/g.
The bioassays for core DH75-106(B) (Table 24), using a two-day-old inoc-
ulum, showed less stimulation than that seen in the other cores. The highest
stimulatory effect was 83%. The pH range was 5.05 to 8.80 and the Pb content
ranged from not detectable to 5.40 ~g/g. Some flasks totally inhibited (100%)
algal growth. Most flasks from sample depth 201.8-233.4 ft showed greater
than 90% inhibition. The pH of these leachates was 2.31 to 4.41 and the Pb
varied from nondetectable to 6.60 ~g/g. Inhibition was observed with leach-
ates of pH range 2.31 to 8.41 and at all measured lead concentrations.
The bioassays of core sample DH76-108 in Table 24 used a six-day-old
inoculum. Stimulation was as great as 411% in the case of the shaking,
autotrophic inoculum with glucose flask of sample depth of 0-18 ft, at a pH
of 7.65 and a Pb content of 1.80 ~g/g. Stimulation was also seen when the
leachate was pH 4.41 to pH 8.50 with Pb content of 1.20 ~g/g to 3.90 ~g/g.
Inhibition was observed in leachates of pH 2.24 to 8.56 and of Pb content
of 1.20 ~g/g to 7.20 ~g/g. The leachate from sample depth 75.8-79.2 ft had
inhibition of 45% to 96%, a pH range of pH 2.24 to pH 6.03 and a lead content
of 1.20 ~g/g to 7.20 ~g/g.
63
-------
Table 26 shows the algal bioassays of core DH75-109, using a two-day-01d
culture for the stationary leachates and a six-day-01d culture for the
shaking leachates. Stimulation was 1413% and 1746% in the shaking, with
glucose flask of both the uninoculated and soil inoculum from sample depth
22.8-38.9 ft, respectively, which had pHis of 8.08 and 6.93 and Pb concen-
trations of 3.60 ~g/g and 2.40 ~g/g, respectively. Leachates showing
stimulation had a pH range of 4.48 and 8.22 and a Pb content range of 0.90
~g/g to 3.60 ~g/g. Inhibition was observed with leachates with a pH range
of pH 4.86 to pH 8.30 and with a Pb range of 0.30 ~g/g to 2.70 ~g/g.
Algal bioassay results of leachates from core DH76-111 are seen in Table
27. A two-day-old culture was used as inoculum for the stationary leachate
bioassays, while a six-day-01d culture was used for the shaking leachate
bioassays. Stimulation was as great as 655% in the bioassays, specifically
the autotrophic and soil inocula of the shaking, with glucose flasks which
had pH values of 7.43 and 6.64 and Pb concentrations of 1.80 ~g/g and 3.90
~g/g, respectively. Inhibition, as seen in all of the flasks of sample depth
102-105 ft, often exceeded 86%. The leachates from sample depth of 102-105
ft had a pH range of pH 1.61 to pH 2.96 and a Pb content range of 1.80 ~g/g
to 10.50 ~g/g. Inhibition was seen from leachates of the pH range, pH 1.61
to pH 7.64 and of Pb content range, 0.90 ~g/g to 10.50 ~g/g.
The algal bioassays of the leachates resulted in stimulation of as high
as 1746% and in inhibition of 100%. The pH and Pb content ranges of the
leachates showing stimulation were pH 3.94 to pH 8.80 and 0.00 ~g/g to 6.00
~g/g. The ranges of pH and Pb content for those leachates showing inhibition
were pH 1.61 to pH 8.56 and 0.00 ~g/g to 10.50 pg/g. The extreme values fdr
a six-day-old culture were 1746% stimulation and 100% inhibition. The ex-
treme values for a two-day-old culture were 297% stimulation and 100% in-
hibition.
The algal bioassays of the low pH leachates, when adjusted to pH 8.0 with
0.1 N NaOH, are seen in Table 28. The leachates were originally pH 2.18 to
pH 5.10 with lead contents from below the detectable limit to 6.00 pg Pb/g.
Raising the pH to 8.0 decreased the toxicity of 42% of the leachates by 30%.
Another 46% of the leachates became less toxic by 1 to 22%. But the remaining
12% of the leachates became more toxic by 36 to 60%.
Relation of Chemical Composition to Presence of Bacteria
After completion of the leaching studies, an attempt was made to corre-
late chemical composition of the cores with presence of iron and sulfur
oxidizing bacteria. Eight samples were selected from cores that had yielded
cultures of these bacteria and six from cores in which these bacteria were
not detected. The samples chosen were not exact replicates of those used for
the bacterial studies since those were no longer available; the samples were
taken from adjacent sections of core. Eight chemicals were determined and the
results are shown in Table 29. Of the two samples with the highest iron con-
tent, one was from a core having iron oxidizing bacteria and one was not.
The same was true for sulfur. Moreover, when the cores were divided into two
groups, one positive for bacteria and the other negative for bacteria, the
mean and standard deviation of each element were not significantly different
64
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Table 28.
Results of algal bioassays of leachates of core samples following pH
adjustment to pH 8.0, percent stimulation or inhibition.~
GS VI-VII medium
Oepth Inoculum with or without Stationary Shaking
Core sample (ft) description o.n (Jlucose leachat~ leachat~
DH75-102 40-50 Uninoculated With -15
Autotrophic -64
inoculum
DH75-104 114.4-131.0 Uninoculated Wi thout +355
Autotrophic -52
inoculum
Soil inoculum -70
Un inoculated With -66
Autotrophic -57
inoculum
Soil i nocul urn -59
DH75-106(B) 178-201.8 Autotrophic With -65
inoculum
201.8-233.4 Uninoculated Wi thout -67 -98
Autotrophic -53 -98
inoculum
Soil inoculum -81 -77
Uninoculated With -98
Autotrophi c -91
inoculum
Soil inoculum -74
DH76-111 102-105 Uninoculated Without -99
Autotrophic -47
inoculum
Soil inoculum -90
Uninoculated With -99
Autotrophic -67
inoculum
Soil inoculum -99
aBioassays with leachates from core samples DH75-102 ~nd ~H75-104 used a six-day-old inoculum,
while bioassays with leachates from core samples DH76-111 and DH75-106(B) used a two-day-old
inoculum.
bInhibition is indicated by a negative (-) value, whereas stimulation is indicated by a
positive (+) value.
65
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Table 29. Chemical analY~iS of overburden core samples in relation to
presence of ba teria which oxidize iron and sulfur compounds.
Chemical Analysis~(~g/g)
Core % CaC03
Sample s~
r~o. (Equiv. )!Y As B Cd Fe Pb .1!L Se
COTes w~th BacteT~a Present
104-4 11.7 1.8 9.5 0.33 439 9.9 0.056 0.21 0.05
104-11 2.0 3.4 5.4 0.36 301 10.9 0.060 0.12 0.03
104-17 12.4 5.1 22.3 0.73 246 9.4 0.213 0.69 1.08
106B-9 1.6 2.2 9.8 0.44 653 13.1 0.066 0.32 0.05
108-1 11.0 8.2 5.1 0.39 769 15.0 0.055 0.38 0.18
108-3 7.1 8.3 5.1 0.48 951 24.2 0.110 0.38 0.16
109-3 12.3 4.0 3.2 0.58 572 6.7 0.025 0.065 0.27
111-1 9.9 5.7 5.9 0.27 485 42.0 0.080 0.16 0.01
-d/ 8.5 4.84 8.29 0.45 552 15.4 0.08 0.29 C.23
x-
s~ 4.47 2.48 6.10 0.15 236.28 11 .62 0.06 0.20 0.36
Cores with Bacteria Absent
102-6 8.7 10.8 7.5 0.52 565 65.5 0.197 0.3 0.33
103-10 1.1 1.4 1.7 0.31 139 7.1 0.060 0.14 0.04
104-6 11.9 2.8 13.9 0.39 504 10.4 0.054 0.20 0.01
104-16 8.1 2.7 3.7 0.38 161 11.0 0.072 0.31 0.11
109-1 9.7 5.0 4.3 0.33 559 10.9 0.050 0.40 0.22
109-2 12.3 8.1 3.6 0.36 1219 15.6 0.042 0.29 0.46
x!Y 8.63 5.13 5.78 0.38 524.5 20.08 0.08 0.27 0.20
s~ 4.06 3.64 4.40 0.07 391.49 22.41 0.06 0.09 0.18
!! All elements are acid digested and reported "Total".
~Method performed: Agriculture Handbook #60 USDA, Method 23C. This value
usually is somewhat high because soil constituents other than li~e ray
react with acid.
c/
- Sulfur reported as percent total content.
!YMean value all samples.
!IStandard deviation.
66
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for the two groups. The table does give additional data supporting the hetero-
geneity of the overburden. It also suggests that such an analysis should
precede leaching studies since some samples had much higher values of poten-
tially toxic elements than other samples. It should be remembered that the
iron and sulfur oxidizing bacteria which were obtained in enrichment culture
in this study were not typical chemolithotrophs and had unidentified nutritional
requirements. For this reason, the lack of correlation of bacteria and chem-
ical analysis of the cores is not too surprising.
67
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SECTION VII
DISCUSSION
The solubilization of metals from mining spoils, including coal mine
spoils, by microorganisms has been well reviewed by Silverman and Ehrlich (1964),
Tuovinen and Kelly (1974), Dugan (1975) and most comprehensively by Brierley
(1978). This leaching of mineral occurs through the oxidation of a substrate,
inorganic or organic. Sulfide minerals produced acid end products (Brierley
1978). Many microorganisms have been studied in relation to their production
of acid and the resulting solubilization of metals. These microorganisms in-
clude the chemolithotrophic Thiobaaillus species (Bryner and Jameson 1958;
Galbraith et al. 1972; Leathen et al. 1953a; 1953b; Temple and Delchamps
1953) and a thermophilic Sulfolobus-like bacterium (Brierley 1978), also
heterotrophs such as the fungus Peniaillium simpliaissimum (Silverman and
Munoz 1971), and the bacterium Baaillus (Tuovinen and Kelly 1974). Hetero-
trophs definitely enhance leaching but at a lower order of magnitude than
chemolithotrophs. Thiobaaillus species play an important role in the solubil-
ization of several metals by greatly accelerating the oxidation of metal
sulfides in mineral and sulfide-bearing coal deposits through acid and ferric
iron production (Silverman and Ehrlich 1964; Temple and Delchamps 1953). The
rate and amount of observable leaching is influenced by the composition of
the deposit, for example, by the amount of pyrite or carbonate present (Dugan
1975; Sokolova and Karavaiko 1964). The toxicity of ground water containing
high acid and/or metal concentrations is therefore dependent upon the overall
composition of the strata through which the ground water percolates (Dugan
1975).
The cursory analysis of the core samples from the West Moorhead coal field
showed a great diversity in the overburden composition. A wide variety of
sedimentary deposits was visually apparent. A diverse chemical composition
was evident from the pH, conductivity, and Pb analyses. A general relation-
ship between the composition of a natural water and the strata with which the
water has been in contact is certainly to be expected (Hem 1970).
The pH values of the core sample slurries encompassed a wide range (pH
2.31 and 9.65 in Tables 10 and 11). The pH values in the water percolating
through the particular zone of each sample would be affected by these
measured values. But ground water, having percolated through many overburden
strata would reflect a composite effect of the different pHis in these strata.
The ground water would therefore not exhibit such a wide pH range; e.g. pH
values range from 5.3 to 8.4 in ground water of the West Moorhead coal field
(unpublished data, U.S. Geological Survey. Billings Division). Localized
areas in the cores demonstrating low pH values, below pH 5.0, could be useful
68
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sources of acidophilic chemoautotrophic bacteria and would be appropriate for
any subsequent leaching study. Low pH values in spoils may be due to the pre-
sence of metal sulfides, pyrite being the commonest, which on exposure oxidize
to the corresponding metal sulfate and sulfuric acid (Fjerdingstad et al. 1976;
Silverman and Lundgren 1959; Temple and Delchamps 1953). This acid production
effectively lowers the pH of the sample and ground water passing through it.
The actual pH depends upon the buffering capacity of minerals in the sample,
the buffering capacity of the water, and the amount of acid produced from the
sample.
Conductivity of the core samples was employed as an easily measured,
general indication of the ion concentration, although conductance can not be
expected to be simply related to ion concentration without a thorough analysis
of the samples (Hem 1970). From the conductivity values observed, one could
postulate that the general inorganic nutrient level in the core would support
a large chemolithotrophic population and be more than adequate for hetero-
trophs (which would be limited by the supply of organic compounds). The con-
ductivity readings could also reflect the acid salts resulting from the neu-
tralization of bacterially produced acid by the carbonates in the overburden.
Lead was tested as an easily measurable parameter of metal composition and
as a possible indicator of autotrophic leaching, not with the idea that high
levels were present. According to the analyses, small quantities of Pb were
present, in the ~g/g of core range, and concentrations were variable in this
range. The solubility of Pb, whether present in the core as a sulfide, hydrox-
ide, or carbonate, would be affected by acid concentration in the leach water.
If leaching in the core samples was due to acid production, lead concentration
in the leachate would be a rough qualitative indicator of this.
Although the West Moorhead coal and the Fort Union coal in general are
composed of low-sulfur subbituminous and lignite coals (Matson and Blumer
1973), considerable variation exists. Chadwick et al. (1975) found vertical
distributional variations of trace elements and sulfur, probably dependent upon
delicate changes in the geological and chemical environment after consolidation
of the coal. Chadwick et al. (1975) also noticed a sulfur enrichment in the
basal footage of the coal seams related to visible concentrations of pyrite
and other metal sulfides in vertical fractures of the coal. The overburden
exhibits even greater variability. Our field observations in the Colstrip
area of the Fort Union coal field show masses of nodular pyrite and also of
large pyrite crystals. These are sporadically distributed in surface spoils
and must reflect a marked variability in pyrite concentration in the over-
burden at that locality. The bicarbonate level of the ground water from over-
burden and coal is relatively high, suggesting high carbonate levels in the
overburden strata of the West Moorhead coal field (unpublished data, U.S.
Geological Survey, Billings Division). The alkalinity of the ground water can
be explained from the low sulfide and/or high carbonate concentrations in the
Fort Union coal deposit and its overburden. There may be no relation between
the amount of metal sulfide leached and metal concentration in the ground
water, due to buffering, neutralizing and precipitation due to carbonates in
the strata and to bicarbonate in the ground water.
Thiobaaillus species are associated with coal mines and coal
ages (Dugan 1975; Tabita et al. 1970). The extremely acidophilic
69
mine drain-
species are
-------
often numerous in coal mines where they grow in drainage channels, on moist
surfaces of pyrite in the coal face and on moist pyrite in the floor and roof
strata after these are exposed to air and water by mining (Dugan 1972; Dugan
1975; Temple and Delchamps 1953). The extremely acidophilic ThiobaciLLus
species include T. thioo~dans and T. ferroo~dans. The spoils, from which
large quantities of acid are produced, have a high sulfur content with pyrite
as the predominant sulfide mineral. When the spoils contain large quantities
of carbonates, the titratable adicity, total salts, sulfate and dissolved iron
in the mine drainage all decrease (Torma et al. 1970). Along with this apparent
decrease in acid production is an increase in population of other ThiobaaiLLus
species, especially T. inter.medius and T. thioparus (Leathen et al. 1953a;
Parker 1947; Tabita et al. 1970). Karavaiko (1961) found ThiobaaiLLus thio-
oxidans in high carbonate sulfur deposits, but not until he analyzed for their
distribution in microzones, instead of in bulk samples. Olson (1978) found
T. ferroo~dans in alkaline coal strip mine drainage in numbers that were equal
to those found in certain acid mine drainages. Studies of strata producing
alkaline drainage indicate the presence of ThiobaciLLus species, which are
associated with acid drainage, but their acid producing activity is masked by
the alkaline composition of the strata (Karavaiko 1961; Olson 1978).
The present study indicated a wide distribution of chemoautotrophic sulfur
and iron oxidizing bacteria probably belonging to the genus ThiobaciLLus, in
the overburden cores from the high carbonate coal fields of eastern Montana
(Tables 12-19). It would be possible to implicate a large number of the
ThiobaciLLus species on the evidence of the percent thiosulfate oxidized and
the resulting pH in the enrichment cultures on the media of Hutchinson et al.
(1965, 1966, 1969). Our results do not implicate the two extremely acidophilic
species. Karavaiko (1959, 1961) found microzones in the high carbonate sulfur
deposit with high populations of T. thioo~dans and with pH values less than
pH 4.0. Unfortunately, the micromethods of Karavaiko (1961) were not employed
in these studies. However, T. ferroxidans was isolated from a revegetation
site (1969-11) at Colstrip, Montana. It oxidized 99% of the ferrous iron and
produced a final pH of 1.84. This isolate was similar to the T. ferrooxidans
cultures isolated from alkaline ground waters of the Decker coal field by
Olson (1978).
Purification of the chemoautotrophic enrichments from the overburden cores
were unsuccessful due to failure of the bacteria to grow adequately. Thio-
sulfate cultures grew on repeated transfers in liquid medium and also on trans-
fers from liquid medium and solid medium to solid medium. However, transfer of
well isolated colonies to fresh solid medium resulted in multiple colony type
formation, while their transfer to liquid medium would not result in growth.
When yeast extract was included in the liquid thiosulfate medium, transfers of
cultures growing in liquid medium produced growth, but transfers from well iso-
lated colonies again did not produce growth.
The purification of the ferrous iron cultures from the overburden cores
was also unsuccessful. The bacteria failed to grow on repeated transfers in
liquid media, even when yeast e~tract, cysteine, glutathione, or IM-MF addi-
tives were included in the iron medium. To place these failures in perspective:
the well described and recognized species of thibacilli grow readily in labora-
tory media. However all researchers on this group encounter strains which
70
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grow only in liquid media, not on agar. Some strains can be subcultured only
in the presence of other organisms. Brierley and others (private communication)
have found strains which respond well to yeast extract, to cysteine and to
other organic supplements but there are still unsolved problems in the culti-
vation of many bacteria with chemolithotrophic properties.
The algal bioassay, following a procedure of the Environmental Protection
Agency (1971), has been used to manage water quality, to evaluate the fertility
of water, and to determine the toxicity of inorganic or organic compounds
(Greene et al. 1975; Payne 1975; Miller et al. 1978). The U.S. EPA (1971),
and Miller et al. (1978) recommend the use of SeZenastrum capricornutum Printz,
a green alga (Chlorophyceae) of the order ChZorococcaZes, as the test alga.
SeZenastrum belongs to the group of ubiquitous algae which include ChZoreZZa,
Scenedesmus, and Ankistrodesmus, which have a wide tolerance toward environ-
mental conditions (U.S. EPA 1978). The parameter used to describe growth of
the test alga is maximum standing crop, which is defined as the maximum biomass
achieved during incubation, but for practical purposes is assumed to be obtain-
ed whenever the increase in biomass is less than 5% per day (U.S. EPA 1971;
Miller et al. 1978). Biomass can be monitored by several methods, which in-
clude dry weight (gravimetric), dry weight (indirect electronic particle
counting), chlorophyll a (in vivo fluorescence, extracted fluorescence and
extracted absorbance), direct microscopic enumeration, and absorbance (tur-
bidity at 750 nm)(U.S. EPA 1971; Miller et al. 1978).
All of the above methods except dry weight were compared to determine
which would be the simplest reliable method under our conditions. The three
methods of monitoring cholorphyll a all reached maximum standing crop, whereas
the other monitoring methods did not. In vivo fluorescence was selected as
the method to measure chlorophyll a, due to the simplicity and rapidity of
the method.
Algal growth curve studies were performed to determine the influence of
pH, glassware acid washing, inoculum size, inoculum age, and inoculum washing
(Fig. 3). These studies were undertaken to improve the reproducibility of the
growth curves in the algal bioassay controls inoculated from six-day-old cul-
tures of the alga (Fig. 4). From the curves seen in Figure 2, glassware acid
washing, inoculum size, and inoculum washing did not affect the maximum stand-
ing crop obtained. All pH values less than 5.3 were toxic to the alga. A two-
or four-day-old inoculum, instead of the six-day-old inoculum, approximately
doubled the maximum standing crop. The transfer of algal cultures on two-,
four-, or even six-day intervals was not consistent with the seven-day interval
recommended by the EPA, but resulted in the production of a more continuously
logarithmic culture which also gave more reproducible results [Fig. 4 (U.S.
EPA 1971, 1978)].
Leaching of the overburden core samples was performed with samples ground
to <80 mesh, or <117 ~m. Particle size influences the rate of leaching (Bryner
and Anderson 1957; Temple and Delchamps 1953). The particle size used was con-
sistent with that used in successful microbiological leaching of copper and
molybdenum minerals [particle sizes ranging from <60 mesh (Bryner and Anderson,
1957) to 63 to 200 ~m (Bosecker et al. 1978)]. There were three inoculum con-
ditions, uninoculated, and autotrophic inoculum, and a soil inoculum. The
71
-------
uninocu1ated flask allowed leaching by the natural bacterial population of the
core samples. The autotrophic inoculum was a mixture of sulfur and iron oxi-
dizing bacteria [as was the inoculum Bosecker et a1. (1978) used in metal
leaching experiments]. The soil inoculum was included to represent the effect
of an actively growing heterotrophic bacterial and fungal contingent. Glucose
was not included in all of the leach media, since organic compounds can have
inhibitory effects on autotrophic growth (Lundgren et a1. 1972). These dif-
ferent incubation procedures were used, not in order to compare them with each
other, but in the hope that one or more of thp.m would yield an interesting
leachate.
The present study used pH and Pb concentration of the leachate to evalu-
ate the leaching in overburden core samples. The 1eachates were also examined
by algal bioassay to determine their potential toxicity. which could be due to
low pH, various metal concentrations, or other factors. The fact that most
1eachates did not show an appreciable pH decrease is almost certainly caused
by the high carbonate concentrations found in the overburden of the West
Moorhead coal field (Matson and Blumer 1973). Some samples did yield acid
1eachates, with pH values as low as 1.61 (Table 27). The Pb values in the
1eachates were roughly similar and not proportional to the Pb content of the
core samples. This strongly implies that autotrophic oxidation did not pro-
ceed at a rate associated with unbuffered high pyrite ores.
Toxicity was observed in the algal bioassays of some 1eachates. Some
1eachates had a stimulatory effect on algal growth, and others had no effect.
This toxicity is readily explained by the low pH in the case of some 1eachates.
However, toxicity was also seen with 1eachates of high pH, as well as with
low pH 1eachates after the pH had been adjusted to 8.0. Lead concentrations
could conceivably influence the observed toxicity, but no relationship between
total soluble Pb concentrations and algal growth inhibition was observed.
Moreover, the Pb concentration in the 1eachates was probably well below toxic
levels and there was a ten-fold dilution of the Pb in the bioassays. Aside
from some acid 1eachates, toxicity was not explained by tne limited analyses
of 1eachates in this study.
Chemoautotrophic bacteria were widely distributed in the overburden cores
from the Bear Creek Study Site of the West Moorhead coal field in the Fort
Union coal deposit located in eastern Montana. This group of bacteria was also
found in the drainage from the Decker coal strip mine, also located in the Fort
Union coal deposit in eastern Montana (Olson 1978). According to our under-
standing of the physiology and nutrition of these bacteria, they do not belong
among the facultative autotrophs but grow only at the expense of an inorganic
energy source. There is an element of uncertainty in this conclusion. But
the odds are that they are producing acid in these core strata, which if true
requires the conclusion that leaching in these overburden cores is accelerated
by this bacterial action. However, in this study most strata did not develop
low pH 1eachates, which, along with the widespread occurrence of chemoautotro-
phic bacteria, means (a) there is a relatively small amount of acid-producing
sulfide present or (b) there is a relatively large amount of neutralizing min-
eral notably carbonates in most strata. This study has not defined which of
these explanations is the case, although Matson and Blumer (1973) show a low
sulfide and a high carbonate level in strata in this area. If toxic elements
72
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are present, their leaching should be accelerated by the action of the chemo-
autotrophic bacteria whose presence has been demonstrated. Toxicity studies
on our leachates, using an algal bioassay, show several effects. Some leach-
ates had a stimulatory effect on algal growth, suggesting an unexplained
eutrophication of the receiving water. Other leachates produced an inhibitory
effect on algal growth which was explained by the low pH of these leachates.
Inhibition, which was reproducible, was observed with other leachates, but
was not explained by the data gathered during this study. All of this sug-
gests that the potential for acid pollution does exist in eastern Montana,
but that its development into a pollution problem would be sporadic and
possibly minor.
73
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SECTION VIII
SUMMARY
Eight test hole cores of overburden grab samples from eastern Montana
coal fields were received from the U.S. Geological Survey. The samples were
visually inspected for evidence of mineralization, aided by an accompanying
geological log for each core. Portions of samples were examined for lead
content, pH, conductivity, and the presence of chemoautotrophic bacteria. The
analyses of core samples, given in Tables 4-11, show quantitatively different
mineralization in the strata and conductivity values varying over a wide
range. These results had no consistent relation to the physical description
of the strata and could not be anticipated by a visual examination of the cores.
The pH values encompassed a much wider range than either Pb or conductivity.
Chemoautotrophic sulfur and iron oxidizing bacteria were isolated from a
number of the core samples. Difficulty was encountered in obtaining pure cul-
tures of these bacteria on solid media. The bacteria differed from other
fastidious iron oxidizers isolated by J. Brierley (private communication).
A culture of iron oxidizing bacteria was isolated from a revegetation
study site (1969-11) at Colstrip, Montana which exhibited evidence of iron
oxidation in close association with coal distributed in the overburden.
This culture was similar to the typical iron oxidizing bacterium,
ThiobaciZZu8 ferrooxidans.
Leaching studies produced a wide range of lead concentrations, pH values,
and conductivity values. Leach media were inoculated with either a soil
mixture obtained from greenhouse pots or a mixture of autotrophic bacteria
obtained from various sources, or were uninoculated. Lead values in the
leachates were roughly similar and not proportional to the Pb content of
the core sample. Autotrophic oxidation did not proceed at the rapid rate
associated with unbuffered high pyrite ores.
Most strata did not develop low pH leachates; a few did (Table 22).
Algal bioassay results (Tables 20-27) showed growth inhibition by some
leachates. The toxicity effect in some cases was explained by low pH values.
Other toxic leachates could not be explained by the data. For the majority of
leachates, no toxicity was observed, instead some leachates produced stimula-
tory effects.
74
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 12. 3. RECIPIENT'S ACCESSION NO.
EPA-600/3-80-046
4. TITLE AND SUBTITLE 5. REPORT DATE
Env i ronmenta 1 Effects of Western Coal Surface Mining Mav 1980 issuina date
Part VII - Microbial Effect on the Quality of Leach Water 6. PERFORMING ORGANIZATION CODE
From Eastern Montana Coal Mine Spoils
7. AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.
Patri ck F. Kimble and Kenneth L. Temple
9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO.
Department of Microbiology I NE625, EHE625
Montana State University 11. CONTRACT/GRANT NO.
Bozeman, Montana 59717 R803950
12. SPONSORING AGENCY NAME AND ADDRESS 13'fYPE fF REPORT AND PERIOD COVERED
Environmental Research Laboratory--Duluth, Minnesota lna
Office of Research and Development 14. SPONSORING AGENCY CODE
U.S. Environmental Protection Agency EPA/600/03
Duluth, Minnesota 55804
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Selected portions of test cores from the overburden of the West Moorhead coal de-
posit in southeastern Montana were examined for possible addition to leach water of
toxic substances and for the presence of iron and sulfur bacteria which might contri-
bute to leaching. Leachates were evaluated by measuring pH, lead, and the effect of
the leachates on the Selenastrum algal assay.
Both sulfur- and iron-oxidizing bacteria were isolated from a number of core
samples. These bacteria differed nutritionally from thiobacilli and from other
bacteria known to be involved in oxidizing sulfur and iron. Concentrations of lead
in the leachates were comparable; they were not proportional to the lead content of
the core samples. Most core samples were nearly neutral in reaction but some were
highly acid. All acid leachates were toxic to Selenastrum. For reasons which were
not determined, some non-acid leachates were also toxic to Selenastrum and some
leachates stimulated Selenastrum growth.
It is concluded that acid formation in overburden spoils would be a problem only
in the circumstance when a potentially acid-forming stratum is so placed during
spoil reclamation that it drains directly into a surface stream. It is recommended
that laboratory bioassays be used adjunct to chemical analysis for identifying
problem strata.
-
17. KEY WORDS AND DOCUMENT ANAL YSIS
a. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Acid formation Energy development
Acid bacteria Coal mining 06/F
Leachate Biological effects 68/0
Toxicity of leachate Acid mine drainage
18. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES
RELEASE TO PUBLIC UNCLASSIFIED 89
20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED
E~ Form 2220-1 lRn.4.-7))
PREVIOUS EDITION IS OBSOLETE
79
(;r U S GOVERNMENT PRINTING OFFICE 1980 -6 57-146/5666
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