Bioremediation by Sulfate Reducing Bacteria of Acid Mine Drainage Paul Frank UC Berkeley Department of Environmental Sciences Abstract Mining activity produces metal sulfide wastes - particularly pyrite - which remain in the mine long after operations cease. When water percolates through a mine where oxygen is present, a series of chemical reactions occur which produce extremely low pH and high concentrations of toxic sulfate and metal ions. This toxic leachate can cause severe aquatic habitat degradation downstream of the mine. This study addresses this environmental problem by harnessing the metabolism of sulfate reducing bacteria, whose ability to reduce sulfate produces carbonate which neutralizes acids and sulfide, which chemically stabilizes toxic metal ions as solid metal sulfides. Batch reactors were set up with mine leachate, bacterial culture, a growth medium, and various sources of organic carbon. Results have shown that bacterial reactions caused copper and zinc reductions of 100%, pH increases of up to 2, and decreases in toxicity of 100%.Introduction Acid mine drainage (AMD), a major environmental hazard that affects aquatic ecosystems around the world, results from the oxidation of metal sulfides, particularly pyrite (FeS2). Discharges from abandoned sulfide ore mines (the most commonly mined sulfides being sulfur, copper, zinc, lead, gold, silver, and uranium) often contain high concentrations of metal sulfides. In lotic environments such as streams, these metal sulfides become rapidly oxidized though chemical and microbial processes (Gray 1998, Bonnissel-Gissinger et al. 1998, Evangelou and Zhang 1995). The resulting sulfate and hydrogen ions lower pH significantly as well as add toxic metal ions to the environment (Gray 1998). Acid mine drainage causes the degradation of aquatic systems through acidification, high concentrations of iron and sulfate, and elevated levels of soluble toxic metals. Under the acidic conditions resulting from AMD, the oxidation of pyrite proceeds by the following reaction (Bonnissel-Gissinger et al. 1998): FeS2 + 14Fe3+ + 8H2O → 15Fe2+ + 2SO42- + 16H+ This reaction demonstrates the polluting capability of the oxidation of pyrite – every mole of pyrite becomes converted to 16 moles of hydrogen and 2 moles of sulfate. This reaction serves as a template for the similar oxidation reactions of most metal sulfides, which also contribute acidity, sulfate, and toxic metal ions to the aquatic environment. Once oxidation has occurred, its products damage the ecosystem in a number of ways. First, the ferric precipitate common to AMD destroys vegetation by blanketing the soil layer and clogging the substrate interstices (Gray 1998). Evidence has also shown that AMD has been responsible for marked declines in aquatic species and ecosystem diversity, as well as productivity. And the toxicity of extreme concentrations of heavy metals and acidity has been linked to total elimination of certain fish species in some aquatic ecosystems (Gray 1998). This study investigated bioremediation as a solution to AMD. In past experimentation, the most common method of combating AMD has been the construction of a treatment wetland downstream from the mine (Machemer et al. 1993; Mitsch and Wise 1997). Such wetlands take up the polluted water, and through microbial processes similar to those used in this experiment purify the water. The wetlands further remove metals through uptake into plant tissues. The drawback of this method is that the problem is not dealt with at the source,but rather after the polluted water has already had an environmental effect. The goal of this project was to determine if sulfate reducing bacteria can be used inside the mine itself such that the leachate is not toxic. Prior research has shown that bacterial metabolism can be of significant use in the removal of metals from wastewater (Dvorak et al. 1992). Sulfate reducing bacteria oxidize simple organic molecules using the sulfate ion as an electron acceptor. This process produces hydrogen sulfide(H2S) and the bicarbonate ion(HCO3-). Hydrogen sulfide readily reacts with heavy metal ions to immobilize the metals as insoluble metal sulfides, while the bicarbonate ions buffer the pH to significantly higher levels (Dvorak et al. 1992). Thus, sulfate is removed as hydrogen sulfide gas and immobile metal sulfides, metals are removed as metal sulfides, and pH is raised, improving water quality. In order to maintain bacterial metabolism, the bacteria must be given both an organic carbon source (as food) and some growth substrate for attachment (the bacteria cannot survive in open water). AMD from an abandoned mine site was treated in batch reactors with an organic carbon source, a growth substrate, and a culture of sulfate reducing bacteria. The mine site lies in the hills east of Oakland, California. Streams originating in the watershed currently show strongly visible signs of habitat degradation such as vegetation loss, low pH, and coating of riparian substrate with oxidized ferric precipitate. It is hypothesized that the bacterial reactions described above will improve this severely toxic water to quality levels similar to unpolluted streams. Methods Study Site All AMD water samples were taken from the Leona Heights Mine, an abandoned sulfur mine in the hills east of Oakland, California. The small creek running out of the mine showed strong visible signs of metal pollution including the characteristic brown-orange coloration of iron precipitate. In addition, toxic levels of several metals and sulfate were found in the creek. Both this creek and the terminal conduit of the watershed, located at the inlet to Lake Aliso several miles downstream in Oakland have been found to be toxic to Ceriodaphnia dubia, a zooplankton species commonly used in aquatic metal toxicity testing. Bacterial Remediation Experiment This study cultured sulfate reducing bacteria in the presence of AMD, an organic carbon source, a substrate on which to grow, and conditionsotherwise similar to those inside a mine shaft. The goal was a determination of whether SRBs can have a significant effect of reducing concentrations and toxicity of metal and sulfate ions while raising pH in water sampled from abandoned mine sites. The bacterial remediation experiment consisted of filling three one-liter sample bottles for each treatment with AMD water from a polluted mine site, a