Autumn 2006
Bioprospecting in the Berkeley Pit
The search for valuable natural products
from a most unnatural world
by Andrea and Don Stierle
Photo courtesy of Lisa Kunkel, The Montana Standard
In addition to the regular staff of scientists and undergraduate
assistants at Montana Tech, the Stierles, at center, have also
worked with local high school and middle school students over
the years on science fair projects focused on Berkeley Pit microbes.
The realization that a compound that could help cure cancer could
be lurking in the Berkeley Pit is thrilling. They like to think
that their microbes could be some of the richest "ore"
ever mined from the Richest hill on Earth.
Most
people think of the Berkeley Pit as a large toxic waste lake,
an unfortunate relic of Butte's proud mining heritage. Don and
Andrea Stierle, however, see the Pit as something more. Like
most of their Natural Products Chemistry colleagues, the Stierles
could be searching the rainforests of Brazil or combing Caribbean
reefs for plants and microorganisms that could yield promising
new drug leads. Instead they are exploring the uncharted expanses
of the Berkeley Pit, which they see as a unique ecosystem with
treasures beyond the vast amounts of copper dredged from this
site for over 25 years.
Anyone living in Butte is probably familiar
with the history of the Pit and its current status as a mine
waste lake. The Superfund strategy will keep the 36 billion gallons
of acidic, metal-rich water from ever escaping the Pit. Until
1995, however, little attention was paid to the biological aspects
of this bleak ecosystem because it was considered too toxic to
support life.
Andrea and Don Stierle set out to change that belief as they
launched a new type of exploration in Berkeley Pit Lake - mining
for microbes. And not just any microbes - they were looking for
microbes that could produce new compounds with real drug potential.
The Stierles
are not new to drug discovery. For the past twenty years they
have looked for anti-AIDS compounds in Bermudian sponge bacteria,
anticancer agents in the bark of redwood trees, and in 1993 found
a fungal source for taxol, an important anticancer compound previously
isolated exclusively from the bark of the elusive yew tree. Andrea
even had the fungus named after her. But they had never before
explored acid mine waste as a source of the next anticancer agent.
Since 1996 the Stierles, and their team of undergraduate researchers,
have isolated and studied a collection of over fifty culturable
bacteria and fungi from one of the more extreme environments
in the lower 48 states.
The Stierles believed that this unusual environment would harbor unusual microbes, which could in turn produce novel chemistry that can be exploited in many ways. The organisms themselves may also be effective bioremediators of the wastewater in which they grow. Their metabolic by-products could have a tremendous impact on the overall ecology of the Pit Lake system by raising the pH of the Pit water, by providing nutrients for other heterotrophs, and by adsorbing metal contaminants. Thus, the research potential of this site is tremendous, and may represent a real renaissance for a geographic area characterized by years of mining, milling, and smelting waste.
The Stierle lab uses a unique tool chest for their "mining venture". Armed with chromatography columns, signal transduction enzyme inhibition assays, a series of antimicrobial testing schemes, and a nuclear magnetic resonance spectrometer assay, they are literally mining this unnatural system for microbes that produce bioactive natural products.
Microorganisms have been an important source of anticancer agents and antibiotics agents of all types since the discovery of penicillin in the 1930's and 40's. Over the years pathogenic microbes develop resistance to widely used drugs and newer more effective antibiotics must be found.
The challenge of Natural Products Chemists like the Stierles is to find new populations of microbes and to effectively isolate compounds with desired biological activity from these organisms. The Stierles have already isolated several exciting new secondary metabolites from the microbial inhabitants of this unusual ecological niche. These compounds include a migraine preventative and several compounds with promising anticancer potential. They have also found an intriguing fungus that appears to pull metals from the Pit water itself.
How would you actually find new bioactive compounds from a Berkeley Pit microbe? It is a complex process. First, the Stierles isolated microbes from water and sediment samples and established them in pure cultures. Each microbe was grown in a series of small liquid culture broths to provide adequate biological material for testing and analysis. This is not an ecological study so the Stierles are not limited to nutrient broths that mimic conditions in the Pit Lake. Instead they use a variety of carbon and nitrogen sources and determine which growth conditions yield the most active natural products. To determine the activity of the compounds produced by their microbes the Stierles must first thoroughly extract each microbial culture using different organic solvents. These extracts are then tested using a series of bioassays or biological tests that can determine if they have potential as antibacterial, antifungal, anticancer, or immune system modulating agents. These tests are used to guide the isolation of pure active compounds from the complex microbial extracts.
Each extract is first tested against a suite of human pathogenic microorganisms, including Staphylococcus aureus, and Streptococcus pneumoniae. In collaboration with Montana State University researcher Allen Harmsen the Stierles are also looking for compounds that show activity against Pneumocystis carinii, causative agent of Pneumocystis carinii Pneumonia, an indicator disease of AIDS patients, and Aspergillus, causative agent of aspergillosis, both of great concern in immunocompromised individuals.
To find compounds with anticancer activity the Stierles use a complex series of signal transduction enzyme assays that identify specific enzyme inhibitors. Inhibition of key enzymes can be an indication that a compound could block the initiation or spreading of cancer cells. In collaboration with University of Montana researcher Keith Parker the Stierles are also looking for compounds with antimigraine activity. The first compound they isolated from their Pit microbe collection showed promise as a migraine preventative.
Looking for
active natural products in this unnatural world has been exciting
and challenging for the Stierle Research Lab. Although their
first four years of work were completely self-funded they have
been able to attract support from the US Geological Survey and
from the National Institutes of Health. Through their funding
they have been able to create new jobs in Butte, hiring two research
scientists and a host of talented undergraduates to help them
with their work. They have also worked with very talented and
hard-working Butte High School students Alexandra Antonioli and
Kels Phelps, and East Middle School student Randi Phelps whose
ongoing Science Fair projects focused on Berkeley Pit microbes.
And Andrea has found that after 26 years at Montana Tech, it
has been nice to actually earn a real salary for all of the work
she does in the lab. But it isn't the funding that keeps the
Stierles looking for new compounds. It is the thrill of discovery,
the realization that a compound that could help cure cancer could
be lurking in the Berkeley Pit. They like to think that their
microbes could be some of the richest "ore" ever mined
from the Richest Hill on Earth.
Can
Algae Clean the Berkeley Pit?
What started off as small experiments
in the laboratory studying Berkeley Pit water in small flasks,
has transformed into a much larger, bench-scale field experiment
using the Berkeley Pit lake as the laboratory and limnocorrals
as giant test tubes suspended in the contaminated water.
For most of the past decade, Dr.
Grant Mitman, a Montana Tech biology professor, has been studying
the ability of algae to remove heavy metal contaminants from
Berkeley Pit water. Through various metabolic, physiological,
and biochemical processes, algae have the potential to reduce
soluble metal ions in acid mine waters. Dr. Mitman, along with
his graduate student, Nicholas Tucci, have applied this potential
bioremediation solution in the Berkeley Pit.
Algae occur naturally in the
Berkeley Pit, but they lack one essential nutrient for growth-nitrate-a
common nutrient found in most fertilizers. If nitrate is added
to pit water, the naturally occurring algae can potentially reach
a concentration of millions of cells per milliliter, a virtual
green soup of suspended organisms that have an ability to permanently
remove dissolved metals from the pit. These organisms have been
used to remediate other pit lakes around the world, and may one
day lead to the natural restoration of the Berkeley Pit.
In the spring of 2004, Mitman
and Tucci deployed nine acid- and metal-resistant cylindrical
limnocorrals along the eastern edge of the Berkley Pit Lake.
Limnocorrals are experimental enclosures which physically isolate
a known volume of water, and allow for the testing of various
experimental manipulations at a relatively low cost. In this
case, 500 gallons of pit water were used to fill the limnocorrals,
and varying concentrations of nitrate were added as the experimental
variable. Each limnocorral, open at the top and closed at the
bottom, measured three feet in width and 10 feet in depth.
Throughout the course of a year, water-quality criteria and algal populations in nutrified limnocorrals were continually monitored and compared with those in non-nutrified limnocorrals to determine if algal growth had an effect on Berkeley Pit water. After the first year of data collection, concentration of algae in the nutrified limnocorrals had increased from undetectable levels to two million cells per milliliter. Additionally, as a result of algal growth, both iron and arsenic concentrations in the pit water were significantly reduced in the nutrified limnocorrals. No significant changes in water-quality or algal growth were detected in the non-nutrified limnocorrals.
The researchers are planning
longer term experiments testing the ability of algae to clean
Berkeley Pit Water. Algae, like any other biological organisms,
need time to achieve a substantial and healthy population. Long
term experiments are necessary to fully determine the bioremediation
potential in the Berkeley Pit.
Spring 2006
Meet
the 2006 Science Fair Award Winners
The Berkeley Pit Education Committee
awarded $50 savings bonds to five grade school students and a
$250 bond to one high school student who competed in the 2006
Montana Tech Science and Engineering Fair. Each of their Fair
projects (see below) explored important topics related to the
Berkeley Pit and mine waste cleanup technologies. Congratulations
to everyone who competed in the Fair, and keep up the good work!
Remember, the Committee will offer awards again at the 2007 Science
Fair, and students are encouraged to choose projects related
to the Pit and mine waste cleanup for next year's competition.
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Water chemistry varies with depth
As reported in all editions of PITWATCH, the water level at the Berkeley Pit has been recorded every month for the past fifteen-plus years. In addition to that monitoring, scientists at the Montana Bureau of Mines and Geology have been sampling and analyzing water from the Berkeley Pit twice a year for its chemical composition and physical properties. Here's an update on some of the results.
In the Berkeley Pit, samples are taken from anywhere between three and nine different depths and analyzed for various dissolved chemicals. Water quality conditions, such as temperature, pH, specific conductance, and dissolved oxygen are also measured at five to 10-foot intervals from the surface to a depth of 300 feet. These same conditions are also measured at a depth near the Pit bottom.
The Berkeley Pit is a chemically
layered system, which means that the chemistry of the water changes
with depth. The brownish-red water at the surface is actually
the least contaminated 
water
in the pit, and the deeper it gets, the worse the water quality.
The color changes as well, going from brownish-red on top to
bluish green at the bottom.
At a certain depth, the chemistry of the water changes so rapidly that it forms a chemical boundary scientists refer to as the chemocline (see illustration and graphs). Water above the chemocline is chemically lighter (i.e., less dense) than the water below. The layering of the two waters is like oil floating on water. The water above the line is also less acidic (higher pH), with lower concentrations of metals. The two layers of water (above and below the chemocline) act like fresh water floating on salt water, never mixing unless stirred. The last time the two mixed was believed to be after the landslide of 1998, when 1.3 to 1.5 million cubic yards of materials sloughed from the southwest wall of the Pit.
Scientists believe the chemical stratification was caused by the many years that the Horseshoe Bend water was diverted into the Berkeley Pit. The water chemistry of the Horseshoe Bend drainage is very different from that of the Pit. Horseshoe Bend water is cleaner and less dense, so it just remained on top, creating the sharp division.
Currently, the Horseshoe Bend flow is diverted to the new water treatment plant and then used for mine operations at the concentrator. As a result, the position of the chemocline keeps changing from year to year.
Tracking the chemocline is important to mine operators. For several years, Montana Resources has operated a process to recover copper from the Berkeley Pit waters. In order to reap the most benefit, the mine needs to extract metals-rich water from below the chemocline, where there's more copper. That dividing line has begun to drift downward in recent years, and as a result, Montana Resources has had to lower the pumps.
In the future, the water chemistry in the Berkeley Pit is expected to change. For example, after extracting copper from the water, the mine circulates the water back into the Pit, and this return water is heavy with iron. Couple that with the fact that the lighter Horseshoe Bend water is no longer entering the Pit, and there will likely be a scientific debate over whether Berkeley will remain a chemically stratified system.
This question and others will only be answered through periodic sampling and continuous monitoring. The chemical changes of the Berkeley Pit will continue to be recorded year after year by the Montana Bureau of Mines and Geology as part of their responsibilities under the 2002 consent decree for this Superfund site.
The Committee thanks Nick Tucci for his help in preparing
this update.