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.
The search for valuable natural products from a most unnatural world
by Andrea and Don Stierle
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.