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p aT rhD ta eani efL iN w e Will algae blo o ming in an acidic, po iso no us Mo ntana mine lead us to an answer fo r Superfund sites? b y Ed win D o b b Fro m t he D e ce mb e r 2000 issue ; p ub lishe d o nline D e ce mb e r 1, 2000

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Pity the sno w geese that settled o n lake berkeley as a sto po ver o ne sto rmy night in No vember 19 9 5. The vast lake, co vering almo st 70 0 acres o f a fo rmer o pen-pit co pper mine in Butte, Mo ntana, ho lds so me 30 billio n gallo ns o f highly acidic, metal-laden water— scarcely a suitable refuge fo r migrating birds stalled by harsh weather. So when the flo ck ro se up and turned so uthward the fo llo wing mo rning, almo st 350 carcasses were left behind. Auto psies sho wed their insides were lined with burns and festering so res fro m expo sure to high co ncentratio ns o f co pper, cadmium, and arsenic. To day o ne need o nly stand o n the viewing platfo rm and lo o k at the pit— the lifeless yello w and gray walls that stretch fo r a mile in o ne directio n and a mile and a half in the o ther and the dark, eerily placid lake— to see that it's ho stile to ward living things. Surely no thing co uld survive these perilo us waters. But in 19 9 5, the same year the birds died, a chemist studying lake co mpo sitio n retrieved so me ro pe co ated with brilliant green slime and to o k it to his co lleagues at Mo ntana Tech o f the University o f Mo ntana, an institutio n lo cals pro udly call the Tech. Having evo lved in partnership with o ne o f the wo rld's richest and lo ngest-running mining districts, it remains a wo rld-class engineering and mining scho o l. Grant Mitman, o ne o f just three full-time bio lo gists o n the faculty, quickly identified the slime as a ro bust sample o f single-celled algae kno wn as Euglena mutabilis. Life had so meho w established an o utpo st in the liquid barren that is the Berkeley Pit.

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Fo r Mitman, finding Euglena pro ved uncannily fo rtunate. At Dalho usie University in Halifax, No va Sco tia, where he received his do cto rate, his passio n was algae. "I trained all my life to be a marine bio lo gist," he says, no ting the iro ny in then having taken a po st at an engineering scho o l in the Ro cky Mo untains. Just as a landlo cked, man-made to xic lake has reunited this scientist and his favo rite subject, so to o has it galvanized the lo ngstanding interest o f chemists Do n and Andrea Stierle, a husband-and-wife team who also wo rk at the university. The Stierles have spent their lives searching fo r naturally o ccurring co mpo unds that can be used in agriculture and medicine. Fo r them, the menagerie o f small o rganisms— mo re than 40 — disco vered in Lake Berkeley during the past five years ho lds much po tential.

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Even mo re impo rtant, perhaps, is the pro mise that so me o f tho se o rganisms can be emplo yed to reclaim the lake— and o ther similar repo sito ries o f mine wastewater— by neutralizing acidity and abso rbing disso lved metals. Beyo nd these po tential benefits are po ssible theo retical advances in bio lo gy. Each new disco very o f a so -called extremo phile— an o rganism adapted to unusually harsh co nditio ns— helps illuminate fundamental bio lo gical pro cesses, fro m metabo lic dynamics to the means and co urse o f evo lutio n, bo th here o n Earth and elsewhere in the universe.

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Lake Berkeley was bo rn o f human appetite and geo lo gical happenstance. During the early 18 8 0 s, just as electricity was lighting up cities and the need fo r co pper mushro o med, an ambitio us pro specto r named Marcus Daly disco vered an eno rmo us depo sit o f the red metal 30 0 feet do wn in his o wn Anaco nda Mine. Fo r the next 50 years, Butte pro vided a third o f the co pper used in the United States and a sixth o f the wo rld's supply— all fro m a mining district o nly fo ur miles square. Thereafter the "Richest Hill o n Earth," as jo urnalists o ften referred to the place, co ntinued to yield vast amo unts o f metals.

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After the Seco nd Wo rld War, the shafts grew deeper— o ne co mpany eventually dug a full mile beneath the surface— but the quality o f the o re diminished. Mining o fficials decided to switch fro m labo r-intensive and dangero us undergro und o peratio ns to o pen-pit mining, a mo re efficient metho d fo r extracting lo w-grade o re. Excavatio n began in 19 55, and so o n the pit became the wo rld's largest truck-o perated mine, alo ng the way displacing so me Italian and Serbo -Cro atian neighbo rho o ds that had gro wn up aro und the o riginal mines o n the east end o f to wn. Mining came to a halt in the early 19 8 0 s, as did the pumps that had been sucking gro undwater o ut o f the mines fo r a century. The flo o ding began. Stro ll acro ss the mining landscape o f Butte to day and yo u will disco ver why the water has had such a pro fo und enviro nmental effect. The land is dull o cher, and the air smells like ro tting eggs. If yo u lo o k clo sely at the waste ro ck, yo u will see pyrite crystals— fo o l's go ld— everywhere. These are all signs o f sulfur. The bedro ck is sho t thro ugh with it. When expo sed to air and water, lo ng-buried sulfide minerals pro duce sulfuric acid, which also helps disso lve o ther minerals fro m surro unding ro ck. Acid-to lerant bacteria that thrive o n iro n and sulfur co mpo unds hasten this pro cess, and when the pumps were shut do wn, the Berkeley Pit became an immense chemical transfo rmer pro ducing ever-greater amo unts o f to xic so up. Making matters wo rse, it's selfperpetuating. By all acco unts, gro undwater will co ntinue to migrate into Lake Berkeley indefinitely. Because o f this threat to the co mmunity, the Enviro nmental Pro tectio n Agency added the pit to the federal Superfund list in 19 8 7. The designatio n also made it part o f the co untry's largest co mplex o f Superfund sites— a series that includes a go o d part o f Butte and the upper 120 miles o f the Clarks Fo rk River watershed. To day Lake Berkeley is the co untry's largest and mo st unusual bo dy o f co ntaminated water. With a pH o f 2.6 , it's as acidic as co la o r lemo n juice. Besides co pper, cadmium, and arsenic, the water co ntains a do zen o ther metals, including aluminum, iro n, manganese, and zinc. But it is precisely because o f these harsh co nditio ns that the lake has caught the attentio n o f life scientists. "We divide the o rganisms we've fo und into two catego ries," says Andrea Stierle. Dressed casually in a T-shirt, jeans, and sneakers, the chemist stands in her lab next to a co unter co vered with petri dishes and Erlenmeyer flasks, each o ne co ntaining a brightly co lo red fungus culture. "The first gro up we call survivo rs," she explains. "They do n't really like the enviro nment, but they put up with it. They're able to defend themselves." Less numero us but far mo re interesting to Stierle and her husband are the dynamic lake inhabitants they call thrivers. Like the survivo rs, these o rganisms arrive by accident— transpo rted by wind and runo ff, depo sited by

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birds, slo ughed o ff bo at bo tto ms o r o ld mine timbers. But unlike their less-prepared co unterparts, they actually flo urish in the presence o f acidity and make use o f so me o f the disso lved metals in the lake. A to xic-waste dump is a bio lo gical haven to thrivers. As they repro duce— fungi require o nly a week to do so , bacteria but a day— characteristics that render them mo re fit beco me widespread. Metabo lic pro cesses are affected. Or as Andrea Stierle puts it, "New enviro nmental niches mean new micro bes, new micro bes mean new chemistry, and new chemistry means new chemical co mpo unds." The stierles have go o d reaso n to believe that the micro bes in Lake Berkeley are a likely so urce o f useful chemicals. Natural co mpo unds have preo ccupied them fo r 20 years, and their partnership has yielded several no table disco veries. The o ne that makes them mo st pro ud o ccurred in the early 19 9 0 s. Then, research sho wed that a substance called taxo l is an effective agent in the treatment o f breast and o varian cancer. In a third o f the wo men receiving taxo l, tumo rs actually shrank. But the news was bittersweet. Taxo l co mes fro m the bark o f Pacific yew trees, a species native to the Pacific No rthwest but nearly extinct. "Ninety-five percent o f them had been cut do wn o r burned as slash," Andrea Stierle says. The few trees left co uldn't pro vide eno ugh taxo l to meet demand. While everyo ne else co ncentrated o n synthesizing the substance and develo ping metho ds fo r gro wing yew trees mo re rapidly, she and Do n fo llo wed a tactic called "bio ratio nal serendipity"— a co mbinatio n o f scientific deductio n and clever, if so metimes pro lo nged, sleuthing. Taxo l might be in yew bark, they reaso ned, because a parasitic o r symbio tic micro be manufactures it there. "Life is everywhere," Andrea says, "and all kinds o f bacteria and fungi are fo und o n plants." Often they pro duce co mpo unds that have never befo re been seen o r explo ited. The best-kno wn example, o f co urse, is penicillin, which was first extracted fro m a mo ld. Fo r almo st two years, Andrea and Do n crisscro ssed the Pacific No rthwest, taking bark samples and, alo ng with plant patho lo gist Gary Stro bel o f Mo ntana State University, testing fo r the presence o f taxo l. Finally, in 19 9 2, in bark fro m a yew tree in Glacier Park, they fo und what they were lo o king fo r— a previo usly unkno wn fungus that pro duces the cancer-killing substance. In ho no r o f Andrea, they named the new o rganism Taxomyces andreanae and applied fo r a patent. Five years later, Bristo l-Myers Squibb purchased the co mmercial rights. The Stierles are using much the same reaso ning to study the bio ta o f the Berkeley Pit. "Whether in defense o r o ffense, every micro be uses its chemistry to pro tect itself," Andrea Stierle explains. In o ther wo rds, bacteria, fungi, and the like manufacture substances that can be po iso no us to o ther micro bes. The generic term fo r such chemicals is "seco ndary metabo lites"— unique co mpo unds that o rganisms assemble fro m the basic building blo cks, o r primary metabo lites, such as carbo n o r hydro gen, that mo st living things ho ld in co mmo n. "It's amo ng the seco ndary metabo lites," Stierle says, "that we find natural pro ducts that can benefit medicine o r agriculture." After finding a pro mising seco ndary metabo lite, the Stierles use standard bio assays to tell whether and to what degree it is to xic. The first is called the brine shrimp lethality test. "It's been fo und that co mpo unds that kill brine shrimp are mo re likely to destro y cancer cells," Stierle says. A seco nd test invo lves E. coli, a co mmo n intestinal bacterium. If the co mpo und repairs DNA in a damaged E. coli, it might also wo rk against cancer. In ano ther assay, the co mpo und is applied alo ng with z a tumo r-causing micro be, to po tato slices. Previo us research has sho wn that if the new chemical pro tects the po tato against tumo r fo rmatio n, it co uld pro ve useful in medicine as well. So far the Stierles have iso lated five no vel co mpo unds, all fro m a single fungus. Each o ne is lethal to brine shrimp. "We've sent them to the Natio nal Cancer Institute fo r further study," Andrea says. Because o f their success with bio ratio nal serendipity, the Stierles fully expect to disco ver many mo re new substances amo ng the o ther fungi and bacteria fro m Lake Berkeley. "We're taking samples everywhere," Stierle says, "fro m the surface, the entire 70 0 -fo o t co lumn o f water, the sediments at the bo tto m." And as time go es by, the o dds impro ve, because with time the thrivers are mo re likely to undergo change. "Under such ho stile co nditio ns the pressure to mutate is intense," Stierle says. "In fact, we may already be seeing the results o f natural selectio n." Clearly the mine flo o d was an enviro nmental disaster with po tentially deadly co nsequences fo r sno w geese and o ther creatures, but it is no w— 18 years and tho usands o f micro bial generatio ns later— pro ving an engine o f evo lutio n. While the stierles watch expectantly, co nfident that co ntingency will yield a chemical bo unty, bio lo gist Grant Mitman is preparing a recipe fo r directing the co mmunity o f life in the Berkeley Pit. So me 3 millio n gallo ns o f gro undwater seep into the lake daily, raising the surface by abo ut o ne fo o t a mo nth. Engineers predict that in abo ut 20 years the water in the pit will rise to the same level as the surro unding gro undwater. Fro m that po int o n, any mo re water that enters the gro und will flo w in the o ppo site directio n. The flo w will reverse co urse, po lluting the alluvial aquifer in the valley belo w the mine and discharging to xic metals into Silver Bo w Creek, the headwaters o f the Clarks Fo rk River. To prevent this calamity, the Atlantic Richfield Co mpany, which is respo nsible fo r Superfund reclamatio n co sts, have to co nstruct a treatment plant befo re the critical level is reached. But the pro cess under co nsideratio n— treating the water with lime, to which metals naturally bind— wo uld pro duce between 50 0 and 1,0 0 0 to ns o f to xic sludge each day. Like many o thers, Mitman believes there is a better way. His way features his favo rite micro bes. "I'm lo o king fo r o rganisms that will clean up the water," Mitman explains. "And I believe algae are the best candidates." Slender, tall, with wire-rim glasses, the 42-year-o ld bio lo gist manages to appear pro fesso rial even as he waxes algal while standing in his walk-in enviro nmental chamber at Mo ntana Tech, ho lding a flask o f light green water in o ne hand and o ne o f dark green water in the o ther. The sign o n the o utside o f the ro o m reads "Gro wth Chamber." It is here that Mitman induces miniature blo o ms in water taken fro m the co ntaminated lake. The emerald blo o m is Euglena mutabilis, the first new resident to be identified; its darker co unterpart is Chlorella ellipsoida vulgaris, o ne o f fo ur o ther algae Mitman has iso lated. Ho lding up the flask o f Chlorella, he says with unmistakable o ptimism, "This is what the Berkeley Pit co uld lo o k like so meday." That green sho uld be the co lo r o f salvatio n might be fitting in a place where so much was sacrificed in the name o f industrializatio n. But behind the symbo lism is a co mpelling bio lo gical argument. Certain algae co nsume metals. Others pro duce bicarbo nate, which reduces acidity. The right o rganisms in the right numbers, the lo gic go es, wo uld help remedy the two mo st no xio us features o f Lake Berkeley. But that is no t the o nly po tential benefit. Algae also co nvert sunlight, carbo n dio xide, and water into sugar. And sugar, Mitman says, "is what makes any system co me alive." It's the fo o d that o ther, larger o rganisms, such as pro to zo ans and fungi, need to survive. So me o f these larger micro bes also reduce acidity. But mo st impo rtant, they co ncentrate metals tenfo ld whenever they co nsume metal-eating algae, a pro cess so metimes referred to as bio lo gical magnificatio n. And when an o rganism dies, it drifts into sediments at the bo tto m o f the lake, where any metals it might co ntain are impo unded. "The key," Mitman says, "is to get the algae go ing first." That's just what he is do ing o n a small scale in his labo rato ry. Under the auspices o f Mo ntana Tech's Mine Waste Techno lo gy Pro gram, Mitman is systematically co nco cting brews o f Berkeley algae. He varies such facto rs as light and temperature, but he's mo st interested in what nutrients each batch receives. Unlike bacteria, pro to zo ans, and fungi, algae feed o n fairly inexpensive and widely available ino rganic nutrients, such as nitro gen and pho spho rus. By do ing no mo re than adding these chemicals, Mitman has been able to trigger an extrao rdinarily rapid gro wth o f algal co lo nies. Iro nically, Euglena, the o rganism that launched the current research pro grams o f bo th the Stierles and Mitman, turns o ut to be highly resistant to metals. It actively excludes them, flo urishing in their presence witho ut making use o f them. "We even grew o ne sample o n a piece o f so lid co pper," Mitman explains. Chlorella has pro ved mo re pro mising. In initial tests, it reduced the mineral co ntent o f the pit water by as much as 10 percent. That may no t seem like much, but Chlorella is o nly o ne o f several indigeno us o rganisms that in all likeliho o d can reduce the lake's to xic co ntents. And as Mitman says, "Every grain o f metal that can be remo ved will save lo ts o f mo ney in the lo ng run." He is no w fo cusing o n ano ther denizen, Chromulina freiburgensis, an alga that has already been sho wn to co ncentrate metals in o ther settings but had never been seen befo re in acidic mine water. Fo llo wing the lab wo rk will co me field tests. Mitman envisio ns barrels flo ating o n Lake Berkeley— each ho using an experimental brew o f o rdinary lake water, algae, and vario us nutrients.

Mitman is co nvinced that ino rganic nutrients can gradually bring abo ut the natural reco very o f Lake Berkeley. Creating a big algal blo o m co uld be as simple as spreading nitrate acro ss the surface o f the water, o r so me fo rm o f mixing may be the best appro ach. So me nitro gen-fixing bacteria inhabit the upper levels o f the lake, he explains, and when they extract nitro gen fro m the air fo r their metabo lic needs, they pro cess nitro gen that o ther o rganisms in the water can use. In large eno ugh numbers, they co uld supply the additio nal nutrients needed to make Lake Berkeley co ntinue blo o ming o n its o wn. "Eventually the system co uld be self-sustaining," Mitman says. Self-sustaining and ever parado xical. Whether bro wnish red due to high iro n-sulfide co ntent o r black because o f metal-co ncentrating algae, Lake Berkeley will remain a fascinating, if fo rbidding, sight— a testament to nature's resiliency as well as a so bering reminder o f the extremes we will go to get the reso urces we want. Life, as Andrea Stierle says, may be everywhere. But it is no t everywhere guaranteed.

Mo ntana Tech maintains a Web site abo ut the Berkeley Pit's enviro nmental issues: mbmgsun.mtech.edu/envberkeley.htm.

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