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Applied and Environmental Microbiology, August 1999, p. 3627-3632, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Seasonal Variations in Microbial Populations and
Environmental Conditions in an Extreme Acid Mine Drainage
Environment
Katrina J.
Edwards,*
Thomas M.
Gihring, and
Jillian F.
Banfield
Department of Geology and Geophysics,
University of Wisconsin
Madison, Madison, Wisconsin 53706
Received 20 October 1998/Accepted 3 March 1999
 |
ABSTRACT |
Microbial populations, their distributions, and their aquatic
environments were studied over a year (1997) at an acid mine drainage
(AMD) site at Iron Mountain, Calif. Populations were quantified by
fluorescence in situ hybridizations with group-specific probes. Probes
were used for the domains Eucarya, Bacteria,
and Archaea and the two species most widely studied and
implicated for their role in AMD production, Thiobacillus
ferrooxidans and Leptospirillum ferrooxidans. Results
show that microbial populations, in relative proportions and absolute
numbers, vary spatially and seasonally and correlate with geochemical
and physical conditions (pH, temperature, conductivity, and rainfall).
Bacterial populations were in the highest proportion (>95%) in
January. Conversely, archaeal populations were in the highest
proportion in July and September (~50%) and were virtually absent in
the winter. Bacterial and archaeal populations correlated with
conductivity and rainfall. High concentrations of dissolved solids, as
reflected by high conductivity values (up to 125 mS/cm), occurred in
the summer and correlated with high archaeal populations and
proportionally lower bacterial populations. Eukaryotes were not
detected in January, when total microbial cell numbers were lowest
(<105 cells/ml), but eukaryotes increased at low-pH sites
(~0.5) during the remainder of the year. This correlated with
decreasing water temperatures (50 to 30°C; January to November) and
increasing numbers of prokaryotes (108 to 109
cells/ml). T. ferrooxidans was in highest abundance
(>30%) at moderate pHs and temperatures (~2.5 and 20°C) in sites
that were peripheral to primary acid-generating sites and lowest (0 to
5%) at low-pH sites (pH ~0.5) that were in contact with the ore
body. L. ferrooxidans was more widely distributed with
respect to geochemical conditions (pH = 0 to 3; 20 to 50°C) but
was more abundant at higher temperatures and lower pHs (~40°C; pH
~0.5) than T. ferrooxidans.
| |
INTRODUCTION |
Acid mine drainage (AMD) has long
been recognized to be greatly impacted by microbial activity.
Iron-oxidizing chemolithotrophs increase the rate of pyrite oxidation
by accelerating the rate-limiting step, the oxidation of
Fe2+ to Fe3+ (16). Ferric iron
oxidizes pyrite by the following reaction: FeS2(s) + 14Fe3+(aq) + 8H2O(l) 
15Fe2+(aq) + 2SO42
(aq) + 16 H+. In low-pH environments, the ferric iron necessary to
drive this reaction is generated primarily by microorganisms because
the rate of ferrous iron oxidation is slow (17). Hence, the
microbial communities involved in sulfide dissolution and acid
generation have received considerable attention for several decades,
due to both the environmental pollution that often results and the economic prospects of bioleaching.
To date, laboratory studies of sulfide mineral dissolution and AMD
production have largely concentrated on the role of two iron-oxidizing
species, Thiobacillus ferrooxidans and, more recently, Leptospirillum ferrooxidans. These organisms are the most
readily cultured from acidic drainage waters and have been found to
greatly accelerate the rate of pyrite oxidation. However, it is well
established that culturing studies, while necessary to assess microbial
physiology, are poor indicators of microbial diversity in situ (1,
11). While molecular studies that circumvent culturing biases in
AMD environments are not numerous, those that have been conducted thus
far largely support the findings of culturing studies: AMD environments
support limited microbial diversity, and most species are readily
obtained via culturing (5, 6, 12). Most molecular studies of
the microbial diversity of AMD environments have been indirect, relying
on DNA extraction, PCR amplification, and cloning techniques. While
these methods are superior to culturing studies as a means of assessing
microbial diversity, they are nonquantitative due to biases associated
primarily with DNA extraction and PCR amplification (4, 13, 14,
18). In situ molecular studies of AMD environments with
oligonucleotide probes are few (3, 15) and thus far suggest
that our understanding of microbial diversity at low pHs is incomplete.
Hence, an understanding of the process of pyrite dissolution and AMD
formation is limited by our understanding of naturally occurring
populations of microorganisms. In particular, the abundance and
distribution of the two species (T. ferrooxidans and
L. ferrooxidans) most commonly isolated from AMD
environments and used for laboratory studies are not well established.
Probing techniques that target whole cells provide quantitative
assessments of environmental microbial populations (for examples, see
references 3, 7, and 15). We used
fluorescence in situ hybridization to evaluate the abundance and
distribution of T. ferrooxidans, L. ferrooxidans,
Archaea, Bacteria, and Eucarya in an
AMD environment as functions of geochemical and environmental conditions. Schrenk et al. have reported probing results for the month
of January (15). Here, we report results for samples
collected over a 1-year period to determine how populations at this
site vary with conditions.
| |
MATERIALS AND METHODS |
Study site.
Iron Mountain is located in the West Shasta
Mining district near Redding, Calif. (Fig.
1). The site is a massive sulfide deposit in rhyolitic host rock that has been mined for the extraction of gold,
zinc, copper, and silver. Mining operations were conducted both
underground and in open pits. Underground mining has resulted in over
10 miles of interconnected tunnels. Mining has also resulted in
fracturing and overall increased permeability of the solid, unprocessed
portions of the deposit. These activities have increased the sulfide
mineral surface area and the total reactive ore that is exposed to
oxygen and surface water.
Conditions at Iron Mountain such as temperature and pH vary
considerably, both spatially and seasonally. Spatially, pH levels
as
low as
3.5 and temperatures in excess of 60°C in some of the
subsurface environments have been reported (
8,
9). Drainage
streams typically have pH values of 2 to 4 and temperatures of
15 to
20°C. Seasonal variations in geochemical conditions result
from
alternating wet and dry seasons, during the winter and summer
months,
respectively. January rainfalls in excess of 35 in. are
common,
contrasting with the little or no rainfall observed from
June through
September.
We compared two environments sampled in January, July, September, and
November: a high-temperature, low-pH environment and
a
lower-temperature, higher-pH environment. At lower pH and higher
temperatures, we examined a disused mine that is in contact with
the
pyrite ore body at a subsurface site known as the Richmond
five-way.
The five-way is located approximately 1,300 ft. into
the mountain and
is a junction between the entrance tunnel and
four tunnels that extend
further into the mountain (Fig.
2).
Temperatures
and pH values vary at the five-way but generally range
from 40
to 50°C and 0 to 1, respectively. The representative sampling
site for the mine environment is an area of the five-way referred
to as
"B-drift" (Fig.
2). The second location is characterized
by higher
pH values and lower temperatures (pH 2 to 4; ~20°C).
For this
environment, we focused on streams peripheral to the
ore body and
drainage in the entrance tunnel to the Richmond five-way
mine. For the
drainage environment, the designation "tunnel" is
used.
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FIG. 2.
Schematic map of the Richmond five-way and entrance
tunnel. Sampling sites are marked with asterisks.
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|
Environmental measurements and sample collection.
Environments within Iron Mountain mine were sampled in January, July,
September, and November 1997. Conductivity and pH measurements were
made with an Orion model 126 conductivity meter and an Orion model 290A
pH meter with an Orion model 9107 temperature-correcting electrode.
Conductivity calibration was performed with a 100-mS/cm reference
solution. Reference solutions of pHs 1, 2, and 4 were used for pH meter calibration.
Water, sediment, and slime samples were collected from each environment
(see above) in sterile 15-ml Falcon tubes and fixed
on-site with 3%
paraformaldehyde in phosphate-buffered saline
(PBS) solution (137 mM
NaCl, 2.7 mM KCl, 4.3 mM Na
2HPO
4, 1.4 mM
KH
2PO
4 [pH 7.4 at 25°C]). Samples were
collected from the same
vicinity within the Richmond mine during each
sampling
trip.
Probe design and specificity.
Oligonucleotide probe design,
sequences, and stringency specifications have been reported previously
(15). Interference of pyrite with the fluorescence in situ
hybridization protocol was tested by hybridization to fixed control
cultures that had been either spotted to (Escherichia coli
DH5
, L. ferrooxidans ATCC 29047, and Aureobasidium
pullulans were obtained courtesy of R. Spear, Department of Plant
Pathology, University of Wisconsin) or allowed to colonize the mineral
surface (T. ferrooxidans ATCC 19859 and Sulfolobus
solfataricus were obtained courtesy of A. Tsang, Department of
Bacteriology, University of Wisconsin).
Hybridization procedure.
Fixed cells in suspension and
pyritic sediment particles were spotted separately at optimal
concentrations of 107 to 109 cells/ml (by
dilution in 70% ethanol) on gelatin-coated [0.25% gelatin and 0.01%
KCr(SO4)2] multiwelled glass slides (10 wells/slide; 10 µl of sample/well) and allowed to dry in a sterile
hood. Pyritic sediments were rinsed once in 70% ethanol prior to being
spotted onto slides to remove unattached cells and debris. The
hybridization procedure followed the protocol of Li et al.
(7).
Samples were examined by epifluorescence and light microscopy with a
Leica DMRX epifluorescence microscope equipped with an
HBO 100-W
mercury arc lamp and red, green, and violet dichroic
filter cubes.
Cells containing DNA in all fixed samples were nonspecifically
stained
with 4',6-diamidino-2-phenylindole (1.5 µg/ml; DAPI; Sigma
Chemical
Co.). Vectashield (Vector Laboratories) was used to prevent
photobleaching. Images were captured with a charge-coupled-device
camera and NIH Image 1.61 software for the Power
Macintosh.
Cell counts were made directly (image analysis software was not used
for counting purposes) by averaging the cell numbers
obtained in a
minimum of 10 fields of view per well from three
to six wells. The
total number of hybridized cells was estimated
by counting the cells
that were hybridized with the probes Bac
338, Arch 915, and Euk 502. All data are shown as the proportion
of cells within a group (domain or
species) relative to the sum
of cells hybridized in all domains. Counts
were made for both
cells in suspension (solutions surrounding
sediments) and cells
attached to pyritic sediment particles.
Calibrations to determine
the volume of material represented in a field
of view and determinations
of the approximate percentage of cells lost
during the washing
protocols were performed experimentally with control
cultures.
| |
RESULTS |
Environmental conditions.
Rainfall records for 1997 were
provided by the treatment plant engineers (Stauffer Management Co.) at
Iron Mountain and have been plotted in Fig.
3. Rainfall in the winter was seasonally high: the site received more than 20 in. of rain in January and more
than 30 in. in February. From February through March, the amount of
rainfall was considerably lower, and rainfall was virtually absent from
June through September. Rain picked up again in November, and the site
received nearly 20 in. of rainfall during that month.
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FIG. 3.
Monthly rainfall totals during 1997 at Iron Mountain.
Data was provided by Stauffer Management Co. and reflects readings
taken at the treatment facility.
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|
Temperature, conductivity, and pH measurements made in the tunnel and
B-drift are shown in Fig.
4. Temperatures
decreased
in both environments throughout the year, while conductivity
rose
during the dry summer months and then decreased in the fall. This
trend was more pronounced in B-drift than in the tunnel. The pH
in
B-drift varied by only 0.22 pH unit over the year. In the tunnel,
the
pH rose almost a unit over the summer and then decreased in
the fall.
Microbial population distribution over time.
The relative
distributions of the three domains, T. ferrooxidans, and
L. ferrooxidans over the course of 1997 in B-drift sediments are shown in Fig. 5. Most notable is the
initial falling proportion of bacteria followed by a rebound, which is
inversely correlated with the rising proportion of archaea followed by
a decline, over the course of the year. Examples of hybridized and
DAPI-stained archaeal cells from B-drift are shown in Fig.
6.
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FIG. 5.
Microbial populations at B-drift for January, July,
September, and November 1997. The numbers used to plot the relative
proportions of cells represent the sum of cells on surfaces and in
suspension associated with the sediments. Cell numbers are normalized
to the sum of the domain counts and thus reflect viable cell
proportions. Error bars reflect standard errors for counting procedures
(see text). Only the lower error bars are shown for stacked data.
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|
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FIG. 6.
Image of probing of archaeal cells in B-drift sediments.
For both the upper and lower sets of images, the cells on the left are
stained with DAPI and viewed under UV fluorescence and the cells on the
right are hybridized with the CY 3 archaeal probe. The upper two images
show cells in suspension, while the lower two show cells adhering to
pyrite sediment surfaces (separated from solution and rinsed with
ethanol prior to probing). Arrows point to clusters of cells, stained
(left) or hybridized (right), that are out of the plane of focus due to
the irregular shape of natural pyrite sediments.
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|
L. ferrooxidans numbers also decreased over the year, while
the number of
T. ferrooxidans organisms rose from
undetectable
to very low levels (Fig.
5). Eucarya were absent in
January and
rose during the dry season. Eukaryotic filaments comprised
the
matrix of slime streamers that developed after the rainy season
in
the Richmond five-way. Prokaryotic cells occurred in high numbers
within the slime streamers as well. Figure
7 shows hybridized
and DAPI-stained
eukaryotic filaments from the five-way.
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FIG. 7.
Example of eukaryotic filaments at the Richmond
five-way. (Left) DAPI-stained cells (viewed under UV); (right)
filaments hybridized with the CY 3 eukaryote probe.
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|
Total cell numbers also varied spatially and seasonally. Cell numbers
in free-flowing water were generally low (<10
5 cells/ml).
In slime streamers and within sediments, they were
considerably higher
(up to 10
8 to 10
9 cells/ml). The number of
cells per milliliter for bacteria, archaea,
and eucarya in B-drift
sediments over the course of 1997 are shown
in Fig.
8. These numbers reflect the total number
of cells that
were hybridized in suspension and attached to sediment
surfaces.
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FIG. 8.
Cells per milliliter for each domain in B-drift
sediments over the course of 1997. Error bars reflect standard errors
for counting procedures (see text).
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|
Microbial population distribution: mine environment versus
drainage.
A previous assessment of the microbial distributions
associated with different sample types in the tunnel and mine
environments (slimes, solutions, and sediments) for January 1997 has
been reported (15). In Fig. 9,
a comparison between the microbial populations in B-drift and the
tunnel, with sample types that dominate the respective environments and
contain the highest cell densities, is shown. A comparison between
completely analogous sample types was not possible, because pyritic
sediments are not present in the tunnel outside of the ore body and
slime streamers are completely absent from the five-way for part of the
year. Data for B-drift hybridizations are for cells associated with
pyritic sediments, while data for tunnel hybridizations are for slime
streamers. The microbial populations shown in Fig. 9 are those for
January and July 1997, which represent the end members' environmental conditions (Fig. 4).
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FIG. 9.
Comparison of microbial populations at the tunnel and
B-drift at the two end-member extremes under environmental conditions
represented in Fig. 4. Patterns are the same as were used for Fig. 5.
Error bars reflect standard errors for counting procedures (see text).
Only the lower error bars are shown for stacked data.
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|
Bacteria dominated both B-drift and the tunnel in January. In the
tunnel,
T. ferrooxidans was abundant and
L. ferrooxidans was present, though in lower numbers. In contrast,
B-drift sediments
lacked
T. ferrooxidans entirely in January
and had low numbers
of
L. ferrooxidans. These general trends
were also seen in July.
While certain microenvironments, such as slime
streamers within
the five-way, have been found to contain high numbers
of
L. ferrooxidans (
15), no sites sampled thus
far within the lower-pH, higher-temperature
environments have been
found to contain significant numbers of
T. ferrooxidans
organisms (
2).
Archaea were not detected in January in either environment; their
numbers rose to high proportions in July, though more significantly
in
B-drift than the tunnel. While eucarya increased in B-drift
during the
summer, they remained undetectable in the
tunnel.
| |
DISCUSSION |
The abundances of eucarya, bacteria, and archaea show seasonal
correlations with geochemical conditions at the Richmond five-way. Spatially, pH influences the prevailing microbial community, as exemplified by the low levels of T. ferrooxidans at sites
where the pH is less than 1 (Fig. 9). However, for B-drift and the
tunnel, these data do not suggest that the more minor, seasonal changes in pH (Fig. 4) have significant effects on the aspects of the microbial
community that we examined. Of the environmental parameters measured in
this study, temperature and conductivity have the strongest impacts on
the prevailing microbial community. Temperature and conductivity vary
as a function of rainfall over the course of the year (Fig. 4), and
these trends mirror many of those seen in microbial distributions. Most
notably, the occurrence of archaea correlates with the rise in
conductivity during the dry summer months, suggesting that the archaeal
species at this site have a competitive advantage under
high-ionic-strength conditions. While the conditions at the Richmond
five-way are extreme relative to those in better-studied AMD
environments, more-acidic, higher-temperature, and higher-metal-load
sites do exist at Iron Mountain deeper in the tunnels (9).
As archaea dominate at the five-way under high-ionic-strength
conditions, it is possible that they may dominate year-round at more
extreme (and less accessible) sites.
The increase in the eukaryote population in the fall correlates with
decreasing temperatures in the mine and with increasing prokaryote
populations. While eukaryotes proportionally increase throughout the
year, their numbers actually decrease in November (Fig. 8) as
prokaryote populations diminish. This is expected, as the decreasing
prokaryote population cannot support as much eukaryotic (heterotrophic) biomass.
The low abundance of T. ferrooxidans at the Richmond
five-way over the year indicates that this species is not a significant member of the microbial community at acid-generating sites (those in
contact with the pyrite ore body). T. ferrooxidans is
abundant only in more moderate-pH (1.5 to 2.3), low-temperature
environments peripheral to the ore body at Iron Mountain. This is
expected, because the pH and temperature conditions at B-drift are
outside of this species' normal growth range (10). In fact,
T. ferrooxidans occurs at detectable levels only when the
temperature drops to within its growth range; its continued low
abundance is likely due to the low-pH conditions that persist. The role
that T. ferrooxidans plays in iron oxidation at this site
occurs after pyrite dissolution and the subsequent acidification of
surface waters. In fact, T. ferrooxidans may play a
beneficial role at this site through the oxidation of ferrous to ferric
iron in drainage streams, as ferric iron precipitates more readily at
higher pHs than ferrous iron. The precipitation of iron plays an
important role in the treatment of contaminated drainage, as toxic
metals, such as arsenic and cadmium, are adsorbed onto the surfaces of precipitates.
L. ferrooxidans is more abundant in extremely low-pH
environments than T. ferrooxidans. However, our sampling has
shown that this species is spatially restricted within the five-way. In
the samples examined, it was found in highest abundance in association with slime streamers and in suspension within the water column. L. ferrooxidans was found to be in lower abundance within
the pyrite sediments and thus may not be the most important
acid-generating species at Iron Mountain.
At all sites examined in this study, T. ferrooxidans and
L. ferrooxidans comprised less than 50% of the viable
microbial population (Fig. 8 and 9). At sites in contact with the ore
body, these species comprised less than 25% of the microbial
population. Numerous studies have explored the rates and mechanisms of
pyrite oxidation by the acidophilic chemolithotrophs T. ferrooxidans and L. ferrooxidans. However, their
applicability to natural systems, in which fluctuating communities of
microorganisms that may or may not include them exist, is unknown. In
this study, we addressed the following questions: what individual
microorganisms and groups of microorganisms are active at sites of acid
generation, and how do they fluctuate in response to environmental
conditions? To our knowledge, this is the first study to quantitatively
track microbial population shifts at an AMD site by molecular
techniques, to show that these shifts are functions of geochemical and
physical parameters. In order to provide the context critical to
understanding the community dynamics involved in acid generation,
further studies are needed to determine the dominant species in AMD
environments as a function of environmental conditions.
| |
ACKNOWLEDGMENTS |
We thank Matthew Schrenk for his input to this study and Brett
Goebel and Norman Pace for assistance with sample collection. We also
thank Iron Mountain Mine Inc. for site access and Stauffer Management
Co. for providing assistance and access to records.
Funding was provided by NSF grant CHE-9521731.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institute, McLean Lab Mailstop #8, Woods Hole, MA 02543. Phone: (508) 289-3620. Fax: (508) 289-2183. E-mail: kedwards{at}whoi.edu.
| |
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Applied and Environmental Microbiology, August 1999, p. 3627-3632, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
