People
have always been intrigued by the unusual biology of underground ecosystems.
Blind fish and albino spiders have long been known to inhabit the deep dark
recesses of caves. Now, we’re beginning to understand that the surface
of Earth, in reality, extends 10,000 feet or more below ground and 10,000 feet
up into the clouds, and is biochemically active everywhere. It is a “catalysphere”
for organisms that feed off of near-surface chemical reactions. A team of geoscientists descended more than 300 feet below ground in the Lackawanna Coal Mine in eastern Pennsylvania, to explore its microbial communities, which play an important role in acid-mine drainage. Photo courtesy of Eleanora Robbins.
Only recently have scientists accepted that acid-mine drainage is a geomicrobial phenomenon. Mining activities expose vast volumes of subsurface rocks and minerals to air and water. Surface disposal of mine waste exposes the sulfur and iron minerals contained in the waste rock to the atmosphere. Both sulfur and iron provide sources of electrons to chemolithotrophic bacteria to “feed” their growth and create acid-mine drainage through a complex web of microbially mediated chemical processes. The release of these contaminants into natural waters, particularly toxic heavy metals, is a widespread problem in areas with a history of coal or metal mining.
Remediation efforts to date have been hampered by insufficient understanding of the geomicrobiology and biogeochemistry surrounding acid-mine drainage formation. The traditional engineering or chemical approaches to acid-mine drainage mitigation, such as pH adjustment via the application of limestone, have generally met with limited success. More importantly, such methods fail to address the root of the problem: the microbial community. Accordingly, it has become increasingly important for scientists to learn more about these communities and the biogeochemical interactions that generate acid-mine drainage in these unusual ecosystems.
We tried a new approach in which we took a multidisciplinary team underground in the Lackawanna Coal Mine, in the anthracite belt of eastern Pennsylvania. Funded and organized by Geovation Technologies, a bioremediation technology company, the project’s primary objective was to further investigate the geomicrobiology and biogeochemistry of an acid-mine drainage pool in the mine. The project team included several geologists, a geochemist, two microbiologists and a mycologist (someone who studies fungi). Each member of the team explored different aspects of the mine ecosystem.
We were particularly interested in the yellow- and orange-colored flocculates of schwertmannite — widely known as “yellow boy.” Commonly associated with the most acidic acid-mine drainage sites, the yellow-boy schwertmannite is a ferric iron oxyhydroxide-sulfate mineral. The yellow ochre formed from dried schwertmannite is considered quite rare and is important in some Native American ceremonies. For several years, Geovation has investigated the use of yellow boy collected from this site as a source of both ferric-iron minerals and microorganisms for the anaerobic bioremediation of extremely persistent environmental contaminants, such as DDT, toxaphene and PCBs.
The study site is protected as part of the Lackawanna Coal Mine Tour in McDade Park near Scranton, Pa. Our tour guide, Tom Supey, actually worked in the mine in his youth, as did his father. Supey’s son is a microbiologist and shares the team’s curiosity about the biology of the mine. Supey took the team and our scientific equipment on a 1,300-foot cable-car trip into the mine, which at a slope of about 25 percent, took us more than 300 feet below ground. We brought down with us two research microscopes to study the microorganisms. Because the mine is a tourist attraction, electricity and outlets were available for both the transmitted and epifluorescent light microscopes. Our setup certainly awed the waves of curious tourists that came by just about every hour. Our study area, however, was away from the tour, in an old shaft that has remained mostly undisturbed since the late 1950s.
Along the floor of the mine, movement grabbed our eyes. A beetle and some albino spiders were moving about. The first pool of standing water was alive with midge flies. Co-author Robbins searched for, but did not observe, the red “wiggle worm” larval stage of these chironomids or their tubes. In some places in the world, when the chironomids metamorphose into midge flies, you have to be careful to cover your mouth and nose. Fishermen in Lake Victoria in East Africa have been killed breathing in these tiny flies during their annual hatching. Midge flies mate in the air and then die. In the Lackawanna Mine, only a few flies were present, but they attracted our interest because we didn’t expect them in a deep mine. The larval-worm stage of these chironomids may be feeding on either the bacterial/archaeal or fungal biomass. Midge worms (sold commercially as fish bait) use hemoglobin as their respiratory pigment; they are bright red where oxygen concentrations in the water are low, and pink where oxygen levels are higher.
Hanging from the roof of the mine were drinking-straw-sized stalactite-like formations, often called “snottites.” One objective was to evaluate whether snottite formation may initiate from hanging fungal hyphae that could serve as focal points for dripping water. This idea came from a 1996 observation by Teresa Rodgers (then at the University of Wisconsin) that some of the snottites were flexible at the Iron Mountain Mine in California, the most acidic place known on Earth. At the Lackawanna Mine, our team found it difficult to decide on the spot if the flexible strings were related to fungal hyphae or spider webs. However, later microscopic evaluation of one snottite by Robbins pointed towards a potential archaeal origin.
The walls near the underground lights were covered with something that looked like green powder. Microbiologist Matt Cottrell of the University of Delaware found that the green came from photosynthetic cyanobacteria that consisted of six-celled filaments. This was consistent with previous findings by National Park Service biologists, who found that underground lights in tourist caves have to be moved around to limit the growth of such cyanobacteria.
Our project may have been the first to take a mycologist underground to investigate an acid-mine drainage ecosystem. Ed Sobek of Microbial Insights, whose specialty is fungal genetics, is the grandson of a coal miner, and his parents still live in the bituminous region of western Pennsylvania. He had not taken two steps off the tourist trail before he began to add new information about the sheer diversity of fungi in the mine.
Every timber
was coated with fungi — white filaments on horizontal wood surfaces and
basidiomycetes with odd fruiting structures lining vertical timbers. The roof
rocks and the anthracite were coated with a fluorescent white mildew. The mine
pool contained long, tube-like masses of fungal rhizomorphs, some of which were
nearly as thick as a pencil. The rhizomorphs were present throughout the mine
pool, and we saw them extend tens if not hundreds of feet beyond the study area
into unexplored portions of the mine shaft. Midge flies and fungal rhizomorphs in an undisturbed layer of “yellow boy” sediment at the bottom of the acid-mine drainage pool at the Lackawanna Mine site. Photo by Bob Zimmer.
Sobek explained that fungi use rhizomorphs as a means of searching for more food, much in the same way plants send out rhizomes to search for moisture and nutrients. In our deepest exploration into the mine shaft, we observed iron bacteria that densely colonize what appeared to be fungal hyphae growing outward from the rhizomorphs and into the acid-mine drainage pool. Microscopic analyses later confirmed such masses of fungal hyphae associated with the rhizomorphs.
Sobek later identified that the fungal rhizomorphs were associated with a species of Armillaria, based on DNA sequencing. Armillaria is a basidiomycete and a member of the so-called white-rot fungi. Such fungi possess some of the most powerful enzymes known for the biodegradation of a wide range of persistent substances, from the lignin found in wood to PCBs.
Co-author Hince has suggested the possibility of a symbiotic relationship between the fungal rhizomorphs and microorganisms in the acid-mine drainage pool — a connection that until now has been largely unrecognized. The fungal rhizomorphs may be deriving nutrients from these mine-pool bacteria in a manner that is roughly analogous to how the fungi in lichen derive nutrients from their photosynthetic, nitrogen-fixing cyanobacteria symbionts.
In addition to exploring the fungi, we also examined the aqueous geochemistry in the mine. Geochemist Chuck Cravotta of the U.S. Geological Survey, Pennsylvania District, measured oxidation-reduction potential, dissolved oxygen and pH to help investigate the oxygen-reduction (redox) “framework” for mineral formation in the pool. Cravotta found lower oxygen levels in a sulfur-rich mine shaft containing a white microbial slime of filamentous bacteria and archaea.
Microscopic examination of the slime showed that the white color was from elemental-sulfur granules associated with the sulfur-oxidizing bacteria Beggiatoa that proliferate at the hydrogen sulfide/oxygen boundary. White filaments of sulfur-oxidizing archaea were found to be even more abundant. Robbins observed that, in contrast to the intracellular sulfur inclusions of the Beggiatoa bacteria, the sulfur granules of the archaeans were outside their cells, among and between their filaments.
Sulfate-reducing bacteria also made their presence known in the usual manner, from the stinky black sediments that were disturbed as we walked along the mine tunnel. Robbins’ microscopic analyses of samples collected from this muck revealed the presence of pyrite octahedrons and framboids commingled with various microbial biofilms. All of these observations attest to the role of microorganisms in the biogeochemical cycling of sulfur species in acid-mine drainage environments.
Our microscopic examinations of samples collected from the low-oxygen mine shaft revealed that large numbers of spirochetes dominated the water-borne bacteria. Recent studies at other acid-mine drainage sites around the world have revealed that spirochetes such as Leptospirillum appear to play an important role in acid-mine drainage formation. Accordingly, the spirochetes found in the Lackawanna Mine may be involved in the initial steps of acid-mine drainage formation at this site as well.
Previous analyses by a Geovation team had revealed the presence of two types of spirochetes in the main acid-mine drainage pool: Magnetospirillum, a magnetotactic bacterium capable of both iron reduction and oxidation; and Azospirillum, a redox-sensing bacterium that can “fix,” or take up atmospheric nitrogen. The findings are highly unusual and unexpected, as both Azospirillum and another nitrogen-fixing microbe identified in the pool, Gluconacetobacter, are diazotrophs — bacteria normally associated with plant crops such as wheat, tomatoes and sugar cane. These nitrogen-fixing bacteria may play a significant role at the base of the overall mine drainage ecosystem by providing a supply of nutrient nitrogen.
The unique findings from the Lackawanna site underscore the need for a better understanding of acid-mine drainage ecosystems and their microbial origins. The multidisciplinary composition and approach of our Lackawanna Mine research team and the results of our collaborative work are a small step in that direction. Before we can develop and implement the next generation of remediation strategies, we need to identify the microorganisms responsible and determine how they are interrelated in these ecosystems in order to understand what conditions trigger the microbial generation of acid-mine drainage. Rather than simply trying to raise the pH or kill the responsible microorganisms (which may be impossible), a better understanding of the geomicrobiology of acid-mine drainage may provide the scientific foundation for more practical and effective remediation strategies.
