Dugan, Patrick R. · The Ohio State University 1975-11 Bacterial Ecology of Strip Mine Areas and...

The Ohio State University

1975-11

Bacterial Ecology of Strip Mine Areas and Its

Relationship to the Production of Acidic Mine

Drainage

Dugan, Patrick R. The Ohio Journal of Science. v75, n6 (November, 1975), 266-279http://hdl.handle.net/1811/22326

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Ohio Journal of Science (Ohio Academy of Science) Ohio Journal of Science: Volume 75, Issue 6 (November, 1975)

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http://hdl.handle.net/1811/22326

BACTERIAL ECOLOGY OF STRIP MINE AREAS AND ITSRELATIONSHIP TO THE PRODUCTION OF

ACIDIC MINE DRAINAGE1-2

PATRICK R. DUG AX

Department of Microbiology, The Ohio State University, Columbus 43210

ABSTRACTDUGAN, PATRICK R. Bacterial ecology

of strip mine areas and its relationshipto the production of acidic mine drain-age. Ohio J. Sci. 75(6): 266, 1975.

The activity of acidophilic bacteria asagents involved in the production of sul-furic acid from iron pyrite (FeS2) foundin association with coal mine refuse orspoils was reviewed. Data was presentedwhich demonstrated the inhibitory effectof anionic detergents and certain organicacids on the growth and metabolism ofthe acidophilic thiobacilli. The influenceof acidic mine drainage on the microfloraof non-acid polluted streams was con-sidered. Also discussed were the hetero-trophic microbes which are indigenous toacid (pH 3.0) streams and acid coal re-fuse, with a section devoted to the po-tential for sulfate reducing bacteria asagents for removal of sulfuric acid fromthe streams.

It has been estimated that 35% of theworld's energy is presently consumed inthe United States; of which 22% is de-rived from coal (Osborn, 1974). This isthe equivalent of 600 to 700 million tonsper year. As of 1971, 40% of the U.S.coal was being extracted via strippingoperations. Two million acres of landhave already been strip mined in orderto achieve this level of production, how-ever, only 0.3% of the stripped land hasbeen reclaimed (Liptak, 1974). The in-centive for stripping coal is economic,with the cost of strip mined coal esti-mated to be $1.25 per ton less than deepmined coal. In Ohio, a considerablyhigher proportion of coal is strip mined

Manuscript received July 22, 1975, revisedNovember 3, 1975 (#75-48).

2Presented as part of a symposium, BiologicalImplications of Strip Mining, held at BattelleMemorial Institute, Columbus, Ohio, onNovember 15, 1974.

(70%) as compared to deep mined (30%).This was equivalent to about 50 milliontons of strip mined coal in Ohio for theyear 1972 (Land Reborn, 1974).

The three general types of mining areschematically illustrated in figures 1A,IB and 1C. Each shows a seam of coallocated under different terrains. If thereis a sufficient depth of rock and soil{overburden) overlying the coal, it wasmore feasible to sink a verticle shaft toreach the coal (fig. 1A). Overburdenusually consists of sandstone, limestoneclay and/or shale which may containother sedimentary deposits such as pyriticminerals. Pyritic minerals and shale areoften found in intimate contact with thecoal or sandwiched between seams of coal.Some of the shale, low grade, non-market-able coal, pyrite and other non-marketableminerals are extracted with the coal andleft on the surface when the coal iscleaned and hauled to market. Thesepiles of minerals are known as refuse pilesand are sometimes referred to as "gobpiles". Several kinds of pyritic mineralsare found in nature, but iron pyrite(FeS2), also known as fool's gold, is theone most commonly encountered in as-sociation with coal.

When the terrain is such that the coalseam outcropped along the side of a slope(fig. IB), it was more expedient to augerinto the hillside. This is known eitheras an auger hole or drift mine. The refusepile is not shown in figure IB but wouldbe present in the area. There is virtuallyno way to segregate pyrite from otherminerals and low grade coal in refuseoriginating from either deep or driftmines.

When a coal seam is sufficiently nearthe surface, a large shovel or drag linecan be used to scoop away the overburdenand expose the seam of coal as illustratedin figure 1C. Shovels with a bucketcapacity of 135 cubic yards and drag-

No. 6 BACTERIA AND ACID MINE DRAINAGE 267

FIGURE 1. Schematic drawing of three general types of coal mining and association of coal withpyrite. (A) deep mining with shaft and refuse pile, (B) auger or drift mine illustratingflow of water drainage, (C) strip mining illustrating rows of overburden spoil banks,high wall.

lines with 220 cubic yards bucket capacityare presently used in Ohio. Once theoverburden has been stripped away, asmaller shovel follows behind and scoopsup the coal seam and loads it into trucks,railroad gondolas, etc. Each strip ofoverburden removed is successively piledbehind the shovel as the shovel "chews"its way along the contour of the hillside.Consequently, overburden which maycontain pyrite is piled in a series of windrow banks separated by a distance equalto the radius of the shovel fulcrum.After stripping in an area is terminated, a"highwall" remains as the result of thefinal cut into the hillside. It is oftenpossible to segregate pyrite from otherminerals in overburden during a strippingoperation and bury it deep in the spoilbank. The term spoil is used in con-junction with stripped overburden, incontrast to the refuse or "gob" from sub-surface mines.

One of the environmental concernsassociated with coal mining results fromthe exposure of pyritic minerals to air(oxygen) and moisture. Exposure of thechemically reduced substance FeS2 tooxygen and water results in oxidation of

the FeS2 to H2SO4 by a complex seriesof chemical reactions which are sum-marized in the following equations:

(1) Fe++- >Fe++++electron

(2)2(SO4

2-+16 electrons+4H+

(3) Sum: FeS2+302+2Ho(2H2SO4+Fe+++

The oxidized iron (Fe3+) formed, sub-sequently reacts with water to produceferric hydroxide and more acid accordingto the following equation:

(4) Fe++++3H2O >Fe(OH)3+3H+

Everyone who has seen drainage frommining regions where pyrite has beenexposed will recognize the yellowishbrown or reddish brown precipitate, oftencalled "yellowboy", that forms on stream-beds. This is the Fe(OH)3 formed inequation (4) and is equivalent to rustedor oxidized iron. Ferric hydroxide alsoreacts with sulfuric acid to form ferrichydroxy sulfate complexes according toequation (5), hence the difference in colorand composition of precipitates depend-

268 PATRICK R. DUGAN Vol. 75

ing upon environmental conditions.Fe(OH)+ may also be present in acidsolution.

(5) Fe(OH)3+2H++SO2- >Fe(OH) (SO4)+2H2O

Figure 2 is a schematic illustration ofthe hydrologic cycle in a refuse pile.Moisture can penetrate all levels of theporous pile and moisture retention, as wellas moisture content, depends upon the

The rate and amount of acid productionwithin the piles is determined by manyfactors such as: the amount of pyrite,particle size of pyrite, presence of micro-organisms which oxidize the pyrite, depthof oxygen penetration, moisture contentof the pile, and temperature range of thepile, and other factors which we presentlydo not understand.

Acid appears to be produced at arelatively consistent rate in a given spoil

Upper Zone StorogeInterception Plut

Depression Storoge PRECIPITATION

i I X X Xmill

Interception

mmEvaporation

ill)))DepressionStorage

X

t T T TTranspiration

BoseflowWater Table

FIGURE 2.

Groundwoter/To DeepStoroge

Schematic illustration of a cross-section of a refuse pile and its hydrology,of Dr. V. Ricca).

(Courtesy

composition of the pile (e.g. the clay,coal, pyrite and sandstone content).Oxygen ordinarily does not penetrateinto the pile depth beyond about 8 to 12inches and is limited by a zone defined asthe oxygen barrier which results fromcompaction of fine sediments (TruaxTrayer Coal, 1971). It is the propensityof refuse piles to produce sulfuric acid(H2SO4) via the oxidation of iron pyrite(or other sulfur containing minerals)(equation 3) which is the primary basis ofour biological concern with strip mining.The sulfuric acid leaches or is flushed outof the pile at a rate determined by localprecipitation and ground water flow(Good et al., 1970; Truax Trayer, 1971).

pile and it can be washed out in a surgeduring a rainfall resulting in a rapid slug-dose of acid entering a drainage or re-ceiving stream. Continued high waterflow due to rainfall will subsequently tendto dilute the acid concentration of thedrainage (Dugan and Randies, 1968;Shumate et al., 1971). Tests with con-trolled sprinkling of refuse piles indicatedthat about 25% of the water applied wasretained in the refuse pile while 75%flushed through almost immediately(Good et al., 1970; Vimmerstedt et al.,1973). Acid loads averaging 305 lbs peracre per day have been shown to beflushed out of refuse piles, and slug loadsof as high as 6.600 lbs/acre/day have

No. 6 BACTERIA AND ACID MINE DRAINAGE 2G9

been recorded. The drainage from suchpiles mav contain as much as: 60,000 mgSO4=/7 (6%), 15,000 mg Fe/7 (1.5%),and 60,000 mg net acidity// (TruaxTrayer, 1971).

It has been calculated that If)5 squaremiles of land across the 11 states thatmake up the Appalachia coal miningregion produce acidic mine drainage,which pollutes 10,500 miles of streams(Acid Aline Drainage in Appalachia,1969). It is estimated that more than370,000 acres of strip mined land will re-quire some form of reclamation in Ohio.Of this total, 180,000 acres are inactiveor abandoned acres that will require amajor reclamation effort at an estimatedcost of $290 million (in 1973) dollars.Abandoned mines discharge over 1 mil-lion lbs of acid per day into Ohiostreams. The acid in the streams ishighly corrosive to bridges, dams andother structures as well as to plumbing.The toxicity and hardness of the waterrestricts its use for irrigational and live-stock watering purposes as well as forrecreational purposes. In general, watercontaminated by acid mine drainageseriously retards virtually all beneficialwater uses at tremendous economic loss(Land Reborn, 1974). The problem willbe intensified by the projected need for a50% increase in coal production by 1980.

MICROBIAL ECOLOGYThe involvement of microorganisms

with acidic mine drainage occurs in atleast 4 ways:

(A) the increased production ofacid via the metabolic activityof the acidophilic Thiobacillusbacteria,

(B) the inhibitory influence of sul-furic acid on the organismsnormally present in receivingstreams,

(C) growth of acid tolerant mi-crobes which will aid in re-covery of acid contaminatedstreams,

(D) the ability of sulfate-reducingbacteria to convert sulfate(e.g. H2SO4) back to sulfidewhich can be precipitated asiron sulfide (FeS).

ACID PRODUCTION BY BACTERIAAcidophilic bacteria of the Thiobacillus-

Ferrobacillus group (i.e. Thiobacillus Ihio-thiooxidans and Thiobacillus ferrooxidans(syn. Ferrobacillus ferrooxidans) ) havelong been recognized as being associatedwith pyrite oxidation and acid productionin coal mine spoils and can be readilyisolated from acid mine drainage water(Duncan el al., 1967; Lorcnz, 19(57;Silverman, 19(54). These are bacteriawhich derive their energy from the oxida-tion of reduced iron (Fe2+) and sulfurcompounds, (both of which are presentin iron pyrite) and derive their cellularcarbon from CO2. The bacteria growoptimally in the pH range of 2.8 to 3.5.Maintenance of an adequate supply ofFe2+ as an energy source in the absenceof high concentrations of organic ma-terial requires an environmental pH lessthan 4.0 because of the rapid auto-oxida-tion of Fe2+ in the presence of O2 abovepH 4.0.

Prior to reports by Colmer and Hinklein 1947 of the involvement of T. ferro-oxidans in the oxidation of pyritic min-erals (Colmer el al., 1949), Carpenter andHenderson (1933) suggested the follow-ing reactions (6 and 7) to explain thetransformation of FeSO4 produced frompyrite:

(6) 4 FeSO4+2 H2SO4+O2 >2 Fe2(SO4)3+2 H2O

(7) 2 Fe2(SO4)3 + 12 H2O >4 Fe(OH)3 + 6H2SO4

In accordance with equation (6), T.ferrooxidans growing on Fe2+ consumesoxygen in the ratio of one mole O2 perfour moles of Fe2+ oxidized to Fe3+ (Leesel al., 1969; Silverman, 1967). The en-ergy being derived from the four electronsliberated from Fe2+. The electrons mustbe accompanied by protons, for whichthere is no biological source, since Fe3+ isnot known to be deposited in the cell,and this may possibly explain the de-pendence of the organism on an acidenvironment. Equation (7) is a non-biological reaction that furnishes an en-vironmental supply of H+. Ferric hy-dioxide will react to form hydroxy sul-fate complexes (equation 9) which havebuffering capacity and can alter equa-


tion (7) and (8) so that less H2O entersthe reactions and less H2SO4 is produced.Equation (6) does not consider the pro-ton requirement for the metabolic reduc-tion of CO2 which is also a requirementof the bacteria.(8) 4FeSO4+O2+10H2O >

4 Fe(OH)3+4 H2SO4

(9) Fe(OH)3+2H++SO2- >Fe(OH) (SO,)+2H2O

The pH optima of all enzymes purifiedfrom T. ferrooxidans are considerablyhigher (i.e.>pH 5.0) (Blaylock and Na-son, 1963; Din et al., 1967; Howard, 1970;Silver, 1968a, 1968b; Vestal, 1971) thanthe environmental pH (i.e. > 4.0), withthe possible exception of the crude prep-aration of the cell envelope associatediron oxidase reported by Bodo and Lund-gren (1974) (optimum pH 2.5 to 3.5).This suggests that the cell envelope ofobligate acidophiles selectively excludeshigh concentrations of H+ from enteringthe cell; a conclusion which has beensupported by a more direct assessment ofthe internal pH of T. ferrooxidans cellswhen the external pH is considerablymore acid (Dewey, 1966). Beck (1960)suggested that T. ferrooxidans has a pas-sive H+ barrier since resting cells do notrespire and are capable of surviving longperiods of storage under acidic conditions.

Although iron pyrite will be oxidizedchemically in the absence of bacteria,ultimately to Fe(OH)3 and H2SO4, thebacteria catalyze the reaction and in-crease the rate of oxidation up to onemillion times the chemical rate (Singer,1970). It has been shown that the ironoxidizing bacteria are more active thanthe sulfur oxidizing bacteria with respectto rates of pyrite oxidation (Leathan et al.,1953). This led to speculation that theprimary role of bacteria in pyrite oxida-tion was the production of ferric ions(equation 1), and that the ferric ionsthus produced oxidized more pyrite withconcommitant regeneration of ferrousions, according to equation (10). The re-cycled iron could again be oxidized by thebacteria and the cycle would continue (Sil-verman, 1967; Singer and Strumm, 1970).

(10) 1415 Fe+++2SQ2

4-Fe S2+8H,(16H+

Singer and Stumm (1970) have con-cluded that under acidic conditions belowpH 4.0, the rate of pyrite oxidation byferric ion is considerably greater than therate of ferrous ion oxidation in theabsence of bacteria. Therefore, the bac-teria must catalyze the oxidation of fer-rous to ferric ion in order to supply theFe+3 to oxidize the pyrite. They con-cluded that the bacterially catalyzedreaction controls the rate of pyrite oxida-tion under acidic conditions. Lau et al.(1970) also came to essentially the sameconclusion in that the bacteria were es-sential to the maintenance of the highferric to ferrous ion ratio, in solution,which is necessary to chemically oxidizepyrite. The mechanism of sulfur oxida-tion by T. thiooxidans would be some-what different in that sulfur is essentiallyinsoluble, therefore requiring direct con-tact of bacterium to substrate (Yogler andUmbreit, 1941).

It should be mentioned that Walshand Mitchell (1972) have been concernedwith the ecological question of how freshlymined spoils initially achieve sufficientacidity to be optimal for the acidophilicThiobacilli. These investigators haveisolated a different microorganism, Me-tallogenium, which also produces someacid via the oxidation of Fe2+ to Fe3+

when accompanied by subsequent hydrol-ysis according to equation 4. The acidproduced creates an environment suit-able for the succession of the Thiobacilli.Although this may be an ecological ex-planation, it need not be a prerequisiteto establishing T. ferrooxidans growthsince chemical oxidation of pyrite wouldaccomplish a lowering of pH to about 4.0over a longer time span.

Much of our knowledge of the microbialoxidation of pyrite has been based uponlaboratory investigations of the activityof isolated or enriched cultures of bacteriaand relatively little data is available oninvestigations in the field. One of themost convincing field studies showingthe activity of bacteria in refuse pileswas carried out by Belly and Brock(1974). These investigators showed astrong correlation between uptake of14CO2 and most probable numbers (MPN)of iron oxidizing bacteria, but not withthe acid tolerant heterotrophic micro-


organisms which were also present in therefuse. They reported maximal 14CC>2uptake in coal refuse 2 to 3 years oldwith only slight incorporation in freshmaterial or material 40 years old. Maxi-mal uptake was always found in samplestaken from the surface above 8 to 10 cmdepth, at temperatures between 20° and3()°C and at moisture content of between23 and 35%, all of which agree with datapreviously published on the basis of lab-oratory investigations (Lau et al., 1970;Truax-Trayer, 1971).

Although actual bacterial catalysis ofpyrite oxidation may occur near the sur-face of refuse piles, the high content ofFe+3 dissolved in acid may leach throughthe spoil banks (See fig. 2) and catalyzethe reaction, shown in equation (10),deep within the pile. Oxidation wouldtake place at a slower rate within the pileresulting in a drainage relatively high inFe+2 which could then be re-oxidized bybacteria in drainage streams or whereveroxygen and CO2 were available.

INHIBITION OF BACTERIALACID PRODUCTION

It has become increasingly apparentthat bacteria play a« major role in theproduction of acidic mine drainage andit has been argued that the most sensibleapproach to the control of mine drainagepollution would be to prevent its forma-tion at the source. One specific suggestionwas to selectively inhibit the acidophilicThiobacilli in refuse and spoil piles(Dugan and Randies, 1968; Dugan, 1972).Studies have been conducted which showthat acidophilic Thiobacilli can be in-hibited under laboratory situations(Dugan and Lundgren, 1964) but thepracticality of inhibition in coal refusepiles has, as yet, not been demonstrated.

It has been reported that the anionicsurfactants, alkylbenzene sulfonate (ABS)and sodium lauryl sulfate (SLS), werevery active inhibitors of T. ferrooxidanswhen cultivated in the laboratory (Duganand Lundgren, 1964). Figure 3A showsthat 5 ppm ABS (approximately 1.4 x10~5M) effectively inhibits iron oxidationby a T. ferrooxidans cell suspension con-taining 4 x 107 cells/ml. Similar dataindicates (fig. 3B) that SLS is an effectiveinhibitor at 2 ppm (approximately 7 x

600

500 -

400

E 300c 600

500

400

300

FIGURE 3.

24 48Time in Hours

72

The effect of inhibitors on iron oxi-dation plotted as increase in Eh byT. ferrooxidans vs. time at 25=I=2OC.(A) alkylbenzene sulfonate (ABS)added at concentrations of 0.5, 2.0and 5.0 mg/liter of culture medium.C is control with no ABS added.(B) sodium lauryl sulfate (SLS)added at concentrations of 0.5, 2.0and 5.0 mg/7 of culture medium.

10-6M). The effectiveness of ABS wasdependent upon the number of bacteriain suspension, as indicated in figures 4Aand 4B where the number of cells insuspension was doubled (2x) and quad-rupled (4x). SLS was not examined inthis manner. Non-ionic detergents andsome sulfonated organic compounds weremuch less toxic to growth than eitherABS or SLS. It was also shown pre-viously by Dugan and Lundgren thatincrease in the hydrogen potential (Eh,a measure of the oxidation/reductionpotential) in this system was correlatedto iron oxidation and to growth of theorganism.

Subsequent laboratory investigationsdemonstrated that several different lowmolecular weight organic acids inhibitediron and sulfur oxidation as well as growth


600

500

400

300

ABS 2 ppm

B

100 r

24 48 72

Time in Hours

FIGURE 4. The effect of alkylbenzenc sulfonale(ABS) on iron oxidation (Eh) byvarious cell densities of T. ferro-oxidans vs. time at 25±2°C; (A) inthe presence of 2.0 mg ABS// and(B) 5.0 mg ABS//. C = controlwith no added ABS. lx = 4 x 107

cells/ml, 2x = 8 x 107 cells/ml, 4x =1.6 xlO8 cells/ml.

of T. ferrooxidans. When a suspension ofT. ferrooxidans (8 mg cell wet weight per5 ml of pH 3.0 H2SO4 which contained5000 Mg Fe2+ per ml as FeSO4 x 7H2O)was aerated in the presence of addedorganic acids at 35°C; the percentage ofinhibition as compared to a control,which did not contain organic additives,was determined after 10 minutes. Figure5 shows the effectiveness of formic, aceticand oxalic acids as inhibitors of ironoxidation by the bacteria and figures 6and 7 show similar data for fumaric,succinic, malic, pyruvic and lactic acids.

Experiments similar to those describedabove (except that mierobial iron oxida-tion was determined by addition of 5.0 mlof 2N HC1 after specified time intervals)

80

60

40

20

• FORMIC ACID

* ACETIC ACID

• OXALIC ACID

10 10" 10" 10" 10"MOLAR INHIBITOR CONCENTRATION

FIGURE 5. Percent inhibition of iron oxidationby washed T. ferrooxidans (8mgccllwet wt/5 ml of solution containing5 mg Fe++ per ml of pH 3.0 H2SO4)in the presence of various concen-trations of formic, acetic, or oxalicacids after 10 min. at 35°C. Valuesare compared to controls in ab-sence of organic acids denned as0% inhibition.

• FUMARIC ACID

* SUCCINIC

• MALIC ACID

10"' 10" 10"'MOLAR

10"CONCENTRATION

FIGURE 6. Inhibition of iron oxidation by T.ferrooxidans due to fumaric, succinicor malic acids. (Experimental data,similar to that shown in fig. 5).

SODIUM PYRUVATE

LACTIC ACID

10' 10"* 10"'MOLAR CONCENTRATION

FIGURE 7. Inhibition of iron oxidation bypyruvic and lactic acids. (Datasimilar to that presented in figs. 5and 6).


provided information on the rate of ironoxidation in the presence of organic acids.Figures 8 and 9 illustrate the effects ofhexanoic and oxalacetic acids, respec-tively, and show that the rate of ironoxidation is markedly retarded by theseorganic acids. In these experiments, ironoxidation was determined by measuringthe absorbance of the Fe+3 + HCl complexat 410 nm.

Other investigators have demonstratedthe inhibitory effects of several organicacids on both T. ferrooxidans and T.

i.O r

0.8

0.6

04

02

NO INHIBITOR

10~3M HEXANOIC ACID

IO"M HEXANOIC ACID

0 2 4 6 8 10 12 14TIME, MIN.

FIGURE 8. Curves showing the decrease inabsorbance at 410 nm due to pro-duction of Fe+3 by T. ferrooxidansin the presence of 10~2M or 10~3Mhexanoic acid vs. time at 35°C.

thiooxidans (Borichewski, 1967) and havealso shown that organic acids are pro-duced by these bacteria (Schnaitmanand Lundgren, 1965). One practical con-sideration which should be mentioned isthat various types of sewage sludge con-tain high percentages of volatile solidswhich have a significant content of or-ganic acids. Addition of sludge to spoilbanks would therefore tend to be in-hibitory to the iron oxidizing bacteriaand of course would add an organic orhumic content to the spoils. Cautionmust be exercised, however, relative tothe presence of viruses, pathogenic micro-organisms and toxic minerals which maybe present in certain sludges.

0.8

0.6

0.4

0.2

• NO INHIBITOR

* 10~eM OXALACETIC ACID

• IO~3M OXALACETIC ACID

4 6 8 10TIME, MIN.

12 14

FIGURE 9. Decrease in absorbance at 410 nmin presence of 10~2M or 10~3Moxalacetic acid. {Data similar tothat presented in fig. 8).

BIOLOGICAL EFFECTS OFACID DRAINAGE

Mine drainage has a deleterious in-fluence on fish, wildlife and plant life inreceiving water (Boccardy and Spaulding,1968; Riley, 1965; Temple and Kohler,1954). Acid drainage also has caused amarked reduction in the microflora ofnon-acid streams and is particularlynoxious to most aerobic and anaerobicheterotrophic bacteria that are indigen-ous to non-acid streams. Acid streamscontain relatively low numbers of acidtolerant aerobic heterotrophic microbeswhich appear to originate in non-acideffluents such as field runoff. The acidtolerant heterotrophs increase in numbersto a maximum of about 2 x 103/ml inacidic streams, until the pH decreases toapproximately 3.0. These organisms thenrepresent the heterotrophic aerobic micro-flora of streams which are comprised of amixture of acid drainage and non-acidwater. A stream composed entirely ofacid drainage does not have a similarmicroflora. Most Gram positive aerobicand anaerobic heterotrophic bacteria die'out very rapidly in acidic water and.comprise a very small percentage of thetotal microbial population. Iron andsulfur oxidizing autotrophic bacteria are

AB

SO

RB

AN

CE

, 4

10

nm

AB

SO

RB

AN

CE

, 41

0 nm


always present in mine water and instreams contaminated by mine water(Tuttle et al., 1968).

The toxic influence on anaerobic growthmay be due to the high hydrogen poten-tial (Eh) of the acid water which resultsfrom high concentrations of oxidized ionsin solution. The adverse influence ofacid drainage on the microflora of non-acid water may be due, in part, to removalof organic nutrients that are essential forheterotrophic growth via acid precipita-tion (Dugan and Randies, 1968; Tuttleet al, 1968; 1969a). If the trophic baseof the ecosystem is depleted, the highertrophic levels will also be proportionatelydepleted.

An attempt was made to assess the re-lative influence of three of the prominentchemical parameters that constitute acidmine drainage (i.e. H+, Fe3+, S0^~), asthey effect the aerobic heterotrophicmicroflora of receiving streams. Theapproach was based on the premise thatphysiological activity of microorganismsis fundamental in triggering the ecologicalsuccession which ultimately results inre-establishment of higher forms of life(e.g. insects, fish, etc.) in the streams.Although iron, sulfate and hydrogen ionsare all characteristic of acidic mine•effluents, they appear to vary indepen-dently with respect to concentration indrainage which originates at differentlocations. It therefore becomes impor-tant to determine which component hasthe most deleterious influence on thebiota; at what concentration the ion isdeleterious; and whether or not significantinteractions exist among ions as they in-fluence biological processes. These con-

siderations have practical signficance inthat they could help to determine levelsof abatement that are essential to allowrecovery of acid polluted streams vianatural processes. It was also desirableto know if a recognizable point exists atwhich growth of a normal aquatic micro-flora is inhibited and/or killed. Thiswould allow prediction of the relativeamount of acid pollution a stream couldtolerate in a variety of circumstances andalso to what extent the water would haveto be treated. Three aerobic hetero-trophic bacteria were isolated and selectedfor study from a non-acid stream (pH 6.2to 8.2) which is located in the immediatevicinity of acid contaminated streams inSoutheast Ohio (Pseudomonas M-l, Fla-vobacterium M-2, Bacillus M-3). Afourth, previously isolated, bacterium:Pseudomonas 229, was also studied.

Although the experimental details arereported elsewhere (McCoy and Dugan,1968), the influence of the three chemicalvariables: pH in the range 2 to 7, ferricion in the concentration range 1 to 100jiig/ml, and sulfate ion in the concentra-tion range 50 to 5000 ng/ml on survivalof each of the bacteria was evaluatedwith the aid of computer analysis. Table1 shows the mathematical models de-veloped which represent the best fitof the four experimental variables. Ba-cillus M-3 was not included becausesufficient data could not be derived fromthe test system for programming. Val-ues which can be predicted on the basisof the models would be expected to varyfrom the experimental values by less than0.2 log unit. Predictions were made onthe basis of the models within the frame-

TABLE 1

Models representing the best fit of the data for three different bacteria.

PSEUDOMONAS Ml

,2-NLOG # BACT. = 32-21 (PH)+4.4(pH)2-0.26(pH)3+0.54 LOG(Fe3+)-0.014(pH)2 LOG(SO|~)

PSEUDOMONAS

LOG § BACT. = 8.2-5.

229

.3(pH)2-0.074(pH)3-0.64LOG(SO|-)+0.21 LOG(Fe3+)-(pH)

FLAVOBACTERIUM M2

LOG # BACT. = 6.4-5.5(pH)+1.4(pH)2-0.084(pH)3+0.12 LOG(Fe3+)

LOG (SO;;-)


work of our stated assumptions. Un-doubtedly we have not considered allsignificant parameters involved in acidicstreams. It was concluded that the testorganisms should be able to grow in anacid stream when the pH is above 5.3, ifiron levels are in the range 1-100 Mg/mland sulfate levels are in the range 50-500 yug/ml.

Growth of the bacteria increased withincreasing iron content provided the pHwas 5 or above. Therefore we probablyshould not be overly concerned with theiron content of waters from a biologicalviewpoint until the concentrations areconsiderably in excess of 100 ng/ml.This comment does not relate to con-siderations such as corrosion and othernon-biological situations. In general,there are complex relationships betweensulfate and pH. That is, sulfate in-fluences growth of the organisms but notin a linear manner. The variable ofparamount importance appears to beH+, with some concern for sulfate.

One might anticipate response to ourgeneralizations by biologists who are in-terested in the welfare of organisms

other than bacteria in streams. Micro-organisms, once growing, will excretemetabolic byproducts into the environswhich have buffering capacity. Theyalso serve as nutrients for other organ-isms. The net result over a period oftime is to re-establish a biological equilib-rium which then includes higher lifeforms. It should also be emphasizedthat this study pertains only to effects ofacid water on the heterotrophic micro-flora of "normal" streams. Our labora-tory also has reported that a high popula-tion of heterotrophic aerobes are indige-nous to certain highly acid (pH 2.5)water (Dugan et al., 1970a; 1970b). Thispopulation of microorganisms representsan ecosystem entirely different from thatfound in the non-acid streams describedabove.

MICROORGANISMS INDIGENOUSTO ACID MINE WATER

An indigenous population of acid toler-ant heterotrophic bacteria is responsiblefor production of slime streamers (figs.10 and 11) in highly acid mine water(pH 2.8). This group of bacteria has not

FIGURE 10. Photograph of the opening of an abandoned drift mine with acid drainage at pH2.5. Height of mine opening is approximately 3 feet.


FIGURE 11. Photograph of "acid streamers" (S) due to bacterial growth in the drainage shown infig. 10. A 3 inch leaf (L) in the water serves as a size comparison.

been well characterized but at least oneisolate has a pH optimum near neutrality(Dugan et al., 1970a; 1970b). This sug-gests that the organisms colonize in thestream creating a localized microcosmthat is different from the surrounding,highly acid environment. An electronmicrograph of a frozen etched preparationof the indigenous slime (fig. 12) showsbacteria embedded within a fibrous ma-trix which is comprised of extracellularpolymer fibrils synthesized by the organ-isms. There appear to be inorganiccrystals precipitated within the fibrousmatrix.

Bohohlool and Brock (1974) have re-ported the growth of a thermophilicmycoplasma, Thermoplasma acidophilum,in refuse piles. Yeasts and filamentousfungi have frequently been isolated fromacidic mine water (Cook, 1966; Dugan,1968; Weaver, 1968). The source oforganic nutrients required to support therather extensive amount of heterotrophicgrowth in mine drainage is not known.One possible source would be from meta-bolic by-products produced by acido-

philic autotrophs. Schnaitman and Lund-gren (1965) reported that organic acidsaccumulate from autotrophic growth ofT. ferrooxidans. Several species of algaehave also been observed in rather largenumbers in acid mine drainage in thepresence of sunlight, especially Euglenaand Ulothrix (Dugan, 1968; Joseph, 1953;Lackey, 1938), which may also enrichthe stream with organic substances.Other sources of heterotrophic nutrientwould be coal and the organic content ofother sedimentary deposits. We alsohave observed the blood worm, Chiro-nomus, living in the slime streamers inacid drainage.

Growth of such a variety of micro-organisms in sulfuric acid environmentsdown to pH 2.0 leads to the conclusionthat our concept of an extreme environ-ment is only relative to that with whichwe are familiar and that this environ-ment is not extreme to many micro-organisms. Conditions are present whichwill promote recovery of acid contami-nated streams to what we consider "nor-mal" if the acid pollution is terminated.


FIGURE 12. Electron micrograph of a frozen etched preparation of the acid slime shown in fig. 11.Bacteria (B) embedded within a matrix of fibrillar strands (F) synthesized by theorganisms can be observed. What appear to be crystals of an inorganic precipitate(IP) embedded within the slime fibrils are also present.

BACTERIAL REDUCTION OFSULFATE TO SULFIDE

Dissimilatory sulfate reducing bacteriaare also capable of growth in acidic minedrainage; provided that sufficient organicmaterial is present to promote consump-tion of oxygen and reduce the Eh of thesystem. These bacteria are obligate an-aerobes and belong to two genera, Desul-fovibrio and Desulfotomaculum. Theyare not acidophilic but are somewhatacid tolerant. Apparently organic andother microparticulates allow a localizedmicrocosm to develop around the cellswhich has a pH higher than that of thebulk solution. Growth of both genera isaccompanied by an increase in pH andreduction of SO= to S=, which is precipi-tated as black FeS in mine water. The

potential utility for microbial sulfate re-duction as an acid mine water abatementprocedure has been described elsewhere(Dugan, 1972, Tuttle et at., 1969a; 1969b).Organic nutrients for this group of bac-teria can be supplied from compostingleaves and other natural detritus, saw-dust, algal growth, sewage sludge, etc.Some caution relative to the use of sewagesludge on refuse piles is indicated, be-cause the high organic content accom-panied by high sulfate concentrations canresult in anaerobic conditions and selec-tion of the dissimilatory sulfate reducingbacteria. This would result in produc-tion of hydrogen sulfide (H2S—a highlytoxic gas with an extremely objectionableodor) which may or may not be pre-cipitated as metallic sulfide (e.g. FeS)depending upon the concentration of


metal ion present relative to the sulfateconcentration (Dugan, 1972).

Acknowledgments.—Portions of this work weresupported by: Grant No. GB5955 from theNational Science Foundation, Grant No. 14-01-0001-805, 980 from the U.S. Office of Water Re-sources Research. Data reported in figures 5through 9 were part of the Ph.D. Dissertationof Dr. Jon Tuttle, Ohio State Univ. 1969. Thefrozen etched specimen (fig. 12) was preparedby Dr. C. H. Remsen.

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