Vol. 53, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2069-20760099-2240/87/092069-08$02.00/0Copyright © 1987, American Society for Microbiology

Algal and Bacterial Activities in Acidic (pH 3) Strip Mine LakesRUTH A. GYURE,' ALLAN KONOPKA,l* AUSTIN BROOKS,2 AND WILLIAM DOEMEL2

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907,1 and Biology Department,Wabash College, Crawfordsville, Indiana 479332

Received 2 March 1987/Accepted 2 June 1987

Reservoir 29 and Lake B are extremely acid lakes (epilimnion pHs of 2.7 and 3.2, respectively), because theyreceive acidic discharges from coal refuse piles. They differ in that the pH of profundal sediments in Reservoir29 increased from 2.7 to 3.8 during the period of thermal stratification, whereas permanently anoxic sedimentsin Lake B had a pH of 6.2. The pH rise in Reservoir 29 sediments was correlated with a temporal increase inH2S concentration in the anaerobic hypolimnion from 0 to >1 mM. The chlorophyll a levels in the epilimnionof Reservoir 29 were low, and the rate of primary production was typical of an oligotrophic system. However,there was a dense 10-cm layer of algal biomass at the bottom of the metalimnion. Production by this layer waslow owing to light limitation and possibly H2S toxicity. The specific photosynthetic rates of epilimnetic algaewere low, which suggests that nutrient availability is more important than pH in limiting production. Thehighest photosynthetic rates were obtained in water samples incubated at pH 2.7 to 4. Heterotrophic bacterialactivity (measured by [14C]glucose metabolism) was greatest at the sediment/water interface. Bacterialproduction (assayed by thymidine incorporation) was as high in Reservoir 29 as in a nonacid mesotrophicIndiana lake.

Acidification of aquatic ecosystems can have profoundeffects on their biological productivity. Most research in thisarea has been concerned with the effects of acid precipita-tion, which has reduced the pH of poorly buffered lakes inNorth America and Scandinavia to 4.5 to 5.5 (27, 30).However, more severe acidification can be found in areaswhere coal is mined. When iron-sulfur pyrites and marca-sites in mine waste are exposed to air and moisture, they areoxidized by chemical and microbially catalyzed reactions tosulfuric acid (32, 35, 37). Total acidity in drainage from coalrefuse can reach 14 g of CaCO3 - liter-', and the pH can dropbelow 2.5 (4).The inputs from acid mine drainage are qualitatively

similar to those found in lakes impacted by acid precipitationbut are quantitatively more severe. As acidic leachate flowsthrough mine spoils and soil, dissolved materials accumulateand are introduced into surface waters. Sulfate concentra-tions may exceed 100 mM, much higher than the levels inseawater (20 mM) or fresh water (0.05 to 0.3 mM) (36, 39).Low pH increases the solubility of iron and aluminum; theirconcentrations in acid lakes may exceed several hundredmilligrams per liter. Other metals, such as copper, zinc, lead,and arsenic, are also present at higher levels than in neutrallakes.These highly acidic lakes, with pH below 4, are uninhab-

itable by organisms making up the higher trophic levels ofaquatic ecosystems. Microorganisms have been found inthese extremely acid environments (2, 8, 15). However, themicrobial processes that occur in acid lakes have not beenstudied systematically to determine whether the range ofprocesses common at neutral pH also proceed at low pH.Some microbial activities can neutralize acidic environ-

ments. The acidity of strip mine lakes decreases with time(4); as a consequence, biological productivity and commu-nity diversity increase. Both theoretical (17) and laboratory(18, 38) studies suggest that anaerobic microbial activity(such as sulfate reduction) can be responsible for acid

* Corresponding author.

neutralization, but these results have not been applied tonatural environments. The activities of these organismsrepresent a biological alternative to chemical neutralizationof acidic lakes, but a detailed understanding of thebiogeochemical cycles in these lakes is necessary to opti-mize the process. The objective of the work described herewas to determine the chemical characteristics and capacityfor organic production and mineralization in two highly acidlakes.

MATERIALS AND METHODSSampling site. Our studies were conducted at the Greene-

Sullivan State Forest (39°00' N, 87°15' W) near Dugger, Ind.This area had been actively strip mined for coal prior to1967, resulting in extensive acid contamination of surfacewaters. The pH of Reservoir 29 was extremely low as aresult of acid leachate from several abandoned coal refusepiles at the northern end. This 225-ha lake is a relativelyshallow basin with a maximum depth of 7 to 8 m at thesouthern end, where an earthen dam separates it from LakeB (Fig. 1). The source of water for Lake B is seepage fromReservoir 29 through the connecting dam. Lake B has asurface area of 20 ha and a highly irregular bottom containingdeep holes (8 to 9 m deep), which remained anaerobicthroughout the study period.

Sampling procedure and field measurements. Water sam-ples were collected with a 2- or 4-liter Van Dorn bottle(Wildco Supply Co., Saginaw, Mich.). For precise samplingat 10-cm intervals, Tygon tubing was lowered to each depth,and water was drawn with a peristaltic pump (Cole-ParmerInstrument Co., Chicago, Ill.). An Eckman dredge was usedto collect profundal surface sediments. These were stored in100-ml polypropylene jars filled to the top to exclude air. Allwater and sediment samples were stored on ice in the darkduring transport and were analyzed within 8 h of collection.Water temperature was measured in the field with a YSIsubmersible thermistor (Yellow Springs Instrument Co.,Yellow Springs, Ohio), and light penetration was measuredwith a Licor submersible quantum probe (Lambda Instru-ments, Lincoln, Nebr.). Oxygen was determined with a YSI

2069

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

acid source

FIG. 1. Map showing relative size and location of Reservoir 29and Lake B. x, Sampling sites.

model 54 oxygen probe (Yellow Springs Instrument Co.),except for hypolimnion samples, which required chemicalassay by use of the permanganate modification of theWinkler technique (1) owing to poisoning of the 02 probe byH2S.Chemical analyses. Sample pH was measured in the field

with a pH meter (Extech Instruments Corp., Waltham,Mass.) and in the laboratory with a pH meter (no. 12;Corning Glass Works, Coming, N.Y.). Total acidity wasdetermined by titrating samples with 0.1 N NaOH; theanaerobic conditions were maintained in hypolimnion sam-

ples by bubbling with N2. Sulfate was measured by precip-itation with BaCl2 (36), and H2S was measured by trappingwith zinc acetate and by subsequent colorimetric determina-tion (5, 14). Total and reduced iron levels were also assayedcolorimetrically; reduced iron was stabilized in field samplesby adding 20 ml of concentrated HCl liter-1 (1). Dissolvedphosphate was assayed by the method of Strickland andParsons (34), and nitrate levels were measured by use ofSzechrome reagents (data sheet no. 239; Polysciences Inc.,Warrington, Pa.). Ammonia was assayed by the indophenolcolorimetric method (1). Sediment porewater was obtainedby centrifugation in sealed serum vials at 6,000 x g for 30min. Porewater was withdrawn with a syringe and assayedby the above methods. The total organic carbon content ofsediments was measured with a carbon determinator (no.WR12; Leco Corp., St. Joseph, Mich.).

Algal biomass. Chlorophyll a and other photosyntheticpigments were assayed by filtering lake water onto glass fiberfilters, extracting with dimethyl sulfoxide and acetone, andmeasuring absorbance at appropriate wavelengths (3). Thevalues were corrected for phaeophytin absorbance (34).

Photosynthetic activity and primary production. A 1- to2-,uCi portion of 14CO2 (specific activity, 40 p.Ci/Iumol;Research Products International Corp., Mt. Prospect, Ill.)was added to 33 ml of lake water in screw-cap test tubes.

These were incubated at 20°C under different light intensitiesfor 2 h. Samples were then filtered onto 0.45-,um-pore-sizemembrane filters (no. GN-6; Gelman Sciences, Inc., AnnArbor, Mich.). The radioactivity on filters was determinedby liquid scintillation counting. Prior to incubation, lakewater was aerated to equilibrate the dissolved CO2 concen-tration with the atmosphere. The data, which describe thephotosynthetic activity of algal populations as a function ofirradiance, were used along with vertical profiles of temper-ature, chlorophyll, and light intensity in a numerical modelto calculate primary productivity (20). The effect of H2S onthe photosynthetic rate was determined by injecting an Na2Ssolution into samples and measuring light-dependent 14CO2incorporation after these additions.

[3lHlthymidine incorporation. Bacterial secondary produc-tion was measured by use of [3H]thymidine (23). Each 15-mlsample of lake water received 0.5 ml of [3H]thymidine(specific activity, 60 to 71 Ci/mmol; Schwarz/Mann, Orange-burg, N.Y.) to a final concentration of 2.5 nM. They wereincubated for 30 min at 18°C in 30-ml serum vials. Forhypolimnion samples, vials were flushed with N2 to maintainanaerobic conditions during incubation. After incubation,samples were filtered through 0.2-pum filters (NucleporeCorp., Pleasanton, Calif.). These filters were placed in 5 mlof ice-cold 5% trichloroacetic acid in 25-ml scintillation vials,incubated on ice for 30 min, placed on a filtering manifold,and rinsed twice more with 3 ml of ice-cold trichloroaceticacid. They were then dissolved in 1 drop of phenethylamine(New England Nuclear Corp., Boston, Mass.) in minivials, 3ml of ACS liquid scintillation fluid (Amersham Corp.,Arlington Heights, Ill.) was added, and samples werecounted. Bacterial production rates were calculated by as-suming that 20% of the cold trichloroacetic acid-extractablelabel was not DNA and using a conversion factor of 2.1 x1018 cells produced. mol of thymidine incorporated intoDNA-1 (28).

[14C]glucose incorporation. The uptake of [14C]glucose wasestimated by measuring both 14C incorporated into particu-late matter and 14C respired as 14CO2 (12). Samples (12 ml oflake water) were incubated in 25-ml tubes fitted with poly-propylene traps containing 0.1 ml of phenethylamine. Tubeswere sealed with red butyl rubber septa, 4 ,uCi of [U-14C]glucose was added, and samples were incubated for 2 hat in situ temperatures. At the end of the incubation, 1.3 mlof ice-cold trichloroacetic acid was added. Samples wereincubated for 2 days to allow 14CO2 to be absorbed into thephenethylamine traps, at which time they were filteredthrough Gelman 0.45-p.m filters. Filters and trapping solutionwere counted after ACS liquid scintillation fluid had beenadded.

RESULTS

Chemical and physical data. Data were collected during1983 to 1985. Although results presented here are primarilyfrom the 1985 sampling season, similar seasonal changeswere found during the other years. Both Reservoir 29 andLake B were thermally stratified during the summer months,with epilimnion temperatures reaching 28°C in July andAugust (Fig. 2). Bottom temperatures remained near 10 to12°C during March to October. Sulfate and total iron con-centrations were higher in the hypolimnion and sedimentporewater than in the epilimnion (Fig. 3).A typical pH profile for each lake during summer stratifi-

cation is shown in Fig. 4. The epilimnetic pH of each lakeremained fairly constant throughout the sampling season;

2070 GYURE ET AL.

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

ACID MINE LAKES 2071

0

CN 3

6

TEMPERATURE ("C)5 10 1520 25 5 10 15 20 25I II

S /~-S~~~~~~~~~~~~~~~~~

S

A B/i~~~~~

FIG. 2. Vertical profiles of temperature on 8 July 1985 in Reser-voir 29 (A) and Lake B (B).

the pH of Lake B (pH 3.2) was slightly higher than that ofReservoir 29 (pH 2.7). Because water entering Lake B mustflow from Reservoir 29 through the earthen dam that sepa-rates them, this difference may be attributed to chemical andbiological transformations that occur during transport,rather than to processes within Lake B itself. The sedimentsand hypolimnetic water of Reservoir 29 were acidic, as werethose in shallow regions of Lake B. However, Lake B was

generally sampled at a deeper site (9 m), at which thesediment and hypolimnion pHs were much closer to neutral-ity. No seasonal changes were observed in the porewater pHof sediment samples from this site in Lake B, but inReservoir 29 there was an increase of 1 pH unit betweenMarch and October 1985 (Fig. 5).

Determination ofpH alone can lead to an underestimate ofthe base-neutralizing capacity of natural waters; aciditytitrations were performed on Reservoir 29 surface water andsediment porewater (Fig. 6). The pH of epilimnion samplesrose quickly when titrated with base, typical of a dilutemineral acid solution with little buffering capacity. The samevolume of sediment porewater, however, required 2.5 timesmore NaOH for neutralization, even though the starting pHwas 1 unit higher. Acidity titrations of anaerobic hypo-

SULFATE (mM)

0

0~% 3

10 20 30 40

2 4 6 8

10 20 30 40

0

2

4

6iZ

a

FIG. 4.(U).

pH3 4 5 6

Vertical profiles of pH in Reservoir 29 (0) and Lake B

limnion water samples were similar to those of sedimentporewater.A gradient of H2S occurred from the sediment through the

hypolimnion of Reservoir 29 during thermal stratification(Fig. 7). Concentrations in excess of 1 mM were measured inthe hypolimnion in August. H2S concentration at thesediment/water interface increased after the initiation ofthermal stratification, as did the pH (Fig. 5). High concen-trations of soluble Fe2+ and sulfide can occur only in acidenvironments. At higher pH they form insoluble FeS. Thiswas observed in Lake B, where the pH in the hypolimnionwas high enough for FeS formation. Free H2S was notdetected in Lake B; soluble Fe21 concentrations in thehypolimnion were 6 to 10 mM. The high concentration ofreduced substances made measurement of oxygen concen-tration problematic when using either an oxygen electrode(Fig. 7) or the chemical Winkler technique. Therefore we

inferred oxygen depletion by the presence of high concen-trations of H2S or Fe2+.

Pi and inorganic nitrogen concentrations were low in theepilimnion (Fig. 8). Reactive phosphate was undetectable(that is, <0.5 ,uM). Ammonia concentrations (1 to 2 ,uM)were much lower than nitrate concentrations (10 ,uM) in theepilimnia of the two lakes. In contrast, nitrate was com-pletely depleted in the hypolimnia, which suggests thatdenitrifying bacteria were active in these anaerobic strata.Ammonia concentrations were highest at the sediment/waterinterface of both lakes and were fivefold higher in Lake Bthan in Reservoir 29.The organic carbon content of Reservoir 29 sediments was

2

1-%

LAJ

S:00.5

2 4 6 8

TOAL IRON (mM)FIG. 3. Vertical profiles of sulfate (U) and total iron (0) in

Reservoir 29 (A) and Lake B (B).

APR MAY JUN JUL AUG SEP OCT NOV1985

4

2

FIG. 5. Changes in pH (-) and H2S (0) concentrations at the

sediment/water interface of Reservoir 29 during 1985.

I , I. . . . . .

*

I I

I I

*

I

I I

1..1I

l*.~ ~ ~E~~~~

II~~~II1/U U

II I,

IUIIiI

. \n~~i~

VOL. 53, 1987

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

10

a

6

4

10

4

4 8 12 16 20 24 28

Na ON added(mmoles)

FIG. 6. Acidity titrations of (A) surface water and (B) sedimentporewater from Reservoir 29. Portions (25 ml) of samples collectedon 19 October 1985 were titrated with 0.1 N NaOH.

13% (wt/wt). High values in acidic environments have beencited as evidence that low pH inhibits decomposition pro-cesses, which leads to an accumulation of organic matter.The organic carbon content of Lake B sediments, whose pHwas more than 2 units higher, was 6.7%.

Biological measurements. Most activity measurementswere made on samples from Reservoir 29, because otherdata (R. A. Gyure, Ph.D. thesis, Purdue University, WestLafayette, Ind., 1986) suggested that seasonal changes inhypolimnion pH were biologically mediated. Algal produc-tivity was determined to assess potential inputs of organicmatter to the sediment. There was adequate light for photo-synthesis through most of the water column. One percent ofthe photosynthetically active radiation present at the lakesurface generally penetrated to a depth of 6.5 to 7.0 m. Thelight extinction coefficient for the water column averaged

OXYGEN (Mg/A)0 2 4 6 8 10

0 I I. I *

2 2

4

6~~~~~~~~~~~~

A~~

2 4 6 8 10

FERROUS IRON (mM). . .

AMMONIA OR NITRATE (AM)a

3-,

15 30 45 15 30 45

FIG. 8. Vertical profiles of ammonia (0) and nitrate (D) inReservoir 29 (A) and Lake B (B) on 23 March 1985.

0.59 during the sampling periods in 1983 to 1985 (n = 29,standard deviation = 0.07).The chlorophyll concentration was used to estimate algal

biomass (Fig. 9; Table 1). The epilimnetic concentrationswere highest in the spring in Reservoir 29. However, withinthe water column the highest concentrations were in thehypolimnion in close proximity to toxic H2S. To determinethe vertical distribution of algae in Reservoir 29 moreprecisely, samples were pumped from 10-cm intervalsthrough Tygon tubing. The highest concentrations occurredfrom samples taken at 6.8 to 7.1 m in Reservoir 29 (Table 2).Chlorophyll concentrations occasionally exceeded 1 mg.liter-'. More than 50% of the algae in the water column werelocated at depths below 6.5 m. Although the H2S concentra-tion at the depth of the chlorophyll maximum was much lessthan near the sediment, values of 30 ,uM were often found.When Lake B was sampled in this manner, a similar layerwas observed, but absorption spectra of extracted pigmentsindicated that bacteriochlorophyll c rather than chlorophylla was the major pigment.

In general, epilimnetic chlorophyll a levels were similar inboth lakes (2 to 6 ,ug of chlorophyll. liter-'). Although the

R.

T-JC)

0 0.5 1 .5 2 25

SUUIDE (rrm)FIG. 7. Vertical profiles of dissolved 02 (with an oxygen elec-

trode) (O), Fe2+ (0), and H2S (A) in Reservoir 29 on 27 August1985.

FIG. 9. Chlorophyll a concentrations (in micrograms per liter) inVan Dorn bottle samples from Reservoir 29 during 1985.

III-- - I

I ~ A I'

*a

*\

1/8 //\* UI I*n U

II I~~~~~0

2072 GYURE ET AL.

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

ACID MINE LAKES 2073

TABLE 1. Chlorophyll a levels in Lake B in 1983

Chlorophyll a level (ig lliter-') on:Depth (m)

23 June 18 July 7 October

0 1.1 0.8 5.71 1.4 1.3 4.62 1.4 1.8 4.53 1.3 2.2 4.84 1.8 3.6 3.75 4.7 3.1 4.86 3.9 4.5 4.57 13.8 10.0 4.08 29.0 26.4 3.29 236.7a 189.4a 58.3a

a Includes bacteriochlorophyll c.

epilimnetic concentrations were relatively low and werecharacteristic of oligotrophic systems, the average chloro-phyll concentration in the water column of Reservoir 29ranged from 10 to 20 p.g. liter-' when the concentratedlayers of pigment were included.

Photosynthetic carbon fixation by phytoplankton samplesfrom the epilimnion and metalimnion was a hyperbolicfunction of irradiance. The maximum photosynthetic rate(Pmax) was never greater than 2 ,ug of C ,ug of chloro-phyll-' h-1 and occurred at irradiance values of about 300microeinsteins * m-2* s- (Table 3). Primary productivity inReservoir 29 for the period 1 May to 31 August 1985 wascalculated by using a numerical model (20) from laboratorymeasurements of photosynthesis and field measurements ofvertical distribution of algal biomass and light (Table 4). Thevalue of 5.3 g of C . m-2 (32 mg of C m-2 day-) fallswithin the range of data for oligotrophic lakes (39). Thephotosynthetic 14CO2 incorporation measurements uponwhich these values were based were performed at CO2concentrations equilibrated with the atmosphere. If in situconcentrations differed from this, the estimate would be inerror. However, 90% of production in the lake was in the top4 m of the epilimnion, where gaseous exchange with theatmosphere can occur.

Photosynthetic activity by samples from below thethermocline was extremely low. A sample from the stratum

TABLE 2. Vertical stratification of algae in the metalimnion ofReservoir 29 dulring 1985

Depth (in) Chlorophyll concna (>g* liter') on:8 July 16 July 19 July 7 Aug 13 Aug 26 Aug

6.5 7 4 4 0 56.6 5 4 4 3 06.7 5 6 5 6 1816.8 16 12 236 117 1 1,4606.9 135 217 480 1,280 193 257.0 646 102 387 48 24 07.1 118 27 127 20 157.2 72 19 157.3 16 12 177.4 26 12 147.5 9 12 IS 0 0

% of totalb 76 50 76 88 62 97

a Concentrations in samples collected by pumping from discrete 10-cmdepth intervals.

b The percentage of total chlorophyll in the water column that was locatedbelow 6.5 m.

TABLE 3. Maximum photosynthetic rates (Pmax) inReservoir 29 during 1985a

Date Sample Pmaxdepth (m) (,ug of C - ,ug of chlorophyll-' - h-1)

5 June 2 0.335 0.20

19 June 2 0.885 0.29

26 June 2 1.345 0.23

10 July 3.1 0.776.3 0.23

24 July 3.1 0.296.3 0.12

a Samples were incubated in the laboratory for 2 h at 20°C at a series of lightintensities, after addition of 1 pLCi of 14CO2.

of highest chlorophyll level had an activity of 0.01 p,g ofC ,pg of chlorophyll-' h-1 at saturating irradiance. Al-though CO2 availability may limit photosynthesis underthese conditions (A. Brooks, W. Doemel, A. Konopka, andR. Gyure, manuscript in preparation), the proximity of thealgal layer to the sulfide-rich hypolimnion also affects pho-tosynthesis. The H2S concentration at the depth of the algalpopulation maximum was generally about 30 ,uM. When H2Swas added to deoxygenated samples from 2 m, a concentra-tion of 1 ,uM inhibited photosynthesis by 50% (Fig. 10).When an equal volume of filtered lake water from 7 m (H2Sconcentration, 750 p,M) was added to samples from 2 m,photosynthetic CO2 fixation was reduced to 0.4% of thecontrol value (2-m samples mixed with an equal volume offiltered 2-m water). However, hypolimnetic water may con-tain toxic substances other than H2S, because the addition offiltered 7-m water which had been sparged with N2 to removeH2S reduced photosynthetic activity to 60% of the controlvalue.The effect of pH on photosynthetic activity was also

examined. Raising the pH caused chemical precipitation,which colored samples and made them turbid. Preventingprecipitation by addition of chelators such as EDTA orsalicylic acid was unsatisfactory, since the necessary con-centrations inhibited photosynthesis. When the pH of sam-ples was adjusted and the described chemical changes wereignored, the rate of photosynthetic 14CO2 uptake was foundto be highest at the in situ pH of 2.6 and decreased at othervalues (Table 5). These data suggest that the algae areadapted to low pH, but other experiments are needed to

TABLE 4. Primary productivity in Reservoir 29 calculatedfor 1 May 1985 to 31 August 1985

ProductivityDepth interval (in) (g of C * m-2)

0-1 ................................... 1.81-2 ................................... 1.42-3 ................................... 0.934 ................................... 0.74 -5 .............................................

0.35-6 .................................... 0.06 -7 .............................................

0.17-7.5 ................................... 0.1

VOL. 53, 1987

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

80

60

40

20

5 10 15

SULFIDE (pM)FIG. 10. Effect of H2S upon photosynthetic 14CO2 incorporation

in deoxygenated 2-m samples from Reservoir 29. Samples wereincubated for 2 h at 25°C and 400 ,mol of photons m-2 s-.

discriminate between the direct effects ofpH and secondaryeffects of other chemical and physical changes.The relative activity of aerobic and anaerobic heter-

otrophs at each depth was assessed by measuring uptake ofradiolabeled organic compounds. Uptake of [14C]glucose(Table 6) indicated that most bacterial activity was concen-trated at the sediment/water interface in Reservoir 29. Incontrast, when bacterial production was determined with[3H]thymidine, values ranged from 7 x 106 to 40 x 106cells * liter-' h-1 through the water column, but there wereno systematic differences between epilimnetic and hypo-limnetic rates.

DISCUSSIONThese two lakes, Reservoir 29 and Lake B, are located in

a poorly vegetated watershed in which the supply of al-lochthonous nutrients for aquatic organisms is probablysmall. Concentrations of dissolved nitrogen and phosphorusin the lakes were very low, and there was little organic inputfrom the sparsely vegetated, barren soil that surrounds thelakes. In the absence of externally supplied carbon, produc-tivity is highly dependent on carbon formed autochthonouslyby photosynthetic organisms. However, primary productiv-ity was also quite low (32 mg of C. m2 day-1), and thusthese acid mine lakes are oligotrophic systems. In compari-son, daily productivity in a eutrophic acidified lake was10-fold higher than in Reservoir 29 during the same period ofthe year (31).The diversity of algae in these lakes was low (Brooks et

al., in preparation), as has been found in other acid environ-ments (6, 21). Chlamydomonas acidophila was the predom-inant alga throughout the water column; Euglena tecta was asignificant component in the deep chlorophyll layer. Super-ficially, the level of algal biomass was also low: chlorophyllconcentrations were usually <3 ,ug. liter-' in the epilim-nion. However, when the concentrated layers in themetalimnion were included, the average chlorophyll level inthe water column was as high as 20 ,ug liter-l, a valueatypically high for an oligotrophic lake (22). The absence ofcrustacean zooplankton in our study lakes may have some

bearing on the high chlorophyll levels. Rotifers were de-tected in both lakes, although no quantitative data were

obtained. Brachionus urceolaris is the most common rotiferreported in extremely acid lakes in the midwestern UnitedStates (24).

TABLE 5. Effect of pH adjustment on net uptake of 14Co2 in2-m samples from Reservoir 29a

pH Amt of 14CO2incorporated (cpm)

1 ...................................... . . . . . 372 .................................... 10,9882.6 ..................................... 20,1363 .................................... 16,0054 .................................... 18,4675 .................................... 10,7906 .................................... 4,2787 .................................... 1,0438 .................................... 760

a Samples were incubated with 2 ,uCi of "4CO2 for 2 h at 19°C and at anirradiance of 350 microeinsteins *m-2 *s-'.

b In situ pH value.

The discrepancy between the amount of algal biomass andthe productivity of the lakes has two bases. First, thespecific photosynthetic rates of the algae were low (Pmax =

0.1 to 1.3 ,ug of C -g of chlorophyll-' h-1). Data onspecific photosynthetic rates in acid systems are scarce.However, in pH-neutral systems, Pmax values of 4 to 6 ,ug ofC g of chlorophyll-1 h-1 are not uncommon (39). Eco-system studies of aquatic systems in the pH range 4 to 5 didnot reveal significant decreases in primary productivityowing to acidification (25, 29). Even in acid lakes nearSudbury, Ontario, Canada, where chlorophytl levels weredirectly proportional to pH, specific photosynthetic rateswere comparable for phytoplankton from lakes at pH 4.4 andpH 6 (21). These results suggest that low pH itself does notreduce photosynthetic activity. The poor nutritional statusof the lakes may have a larger effect.

Nutrient availability does influence photosynthetic rates;rates are depressed in nitrogen- or phosphorus-limited cul-tures of phototrophs (19). Another limiting factor in theseacid lakes may be C02, since at low pH there is no pool ofbicarbonate alkalinity, so that the flux of CO2 is dependenton atmospheric diffusion and equilibration in the watercolumn.The vertical distribution of phytoplankton is a second

factor which lowers the productivity of these systems. Mostof the phytoplankton is located at depths where irradiance is<2% of the surface irradiance. Furthermore, the proximityof this algal layer to the sulfide-rich hypolimnion reduces itsphotosynthetic activity, because of sulfide toxicity.The location of the algal chlorophyll maximum in relation

to the H2S gradient is surprising. H2S concentrations of 30p.M were found in this stratum; photosynthesis of epilim-

TABLE 6. Metabolism of [14C]glucose by water samplesfrom Reservoir 29ao% [14C]glucose metabolized on:

Depth (in)July 27, 1983 June 5, 1984 June 17, 1984 July 24, 1984

0 0.095 0.0121 0.0672 0.083 0.039 0.0133 0.0814 0.0975 0.270 0.013 0.0237 2.200 0.534 0.045 0.8637.5 2.600 0.248

a Samples (12 ml) were incubated for 2 h with 20 ,Ci of ['4C]glucose at insitu temperature.

2074 GYURE ET AL.

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

ACID MINE LAKES 2075

netic algae was severely inhibited by H2S concentrationsone-fifth this value. The Pmax of samples from the chloro-phyll maximum was 30- to 100-fold lower than in epilimneticsamples; yet when natural samples from the chlorophyllmaximum were examined microscopically, motile, morpho-logically intact cells of C. acidophila and E. tecta werefound.The occurrence of this layer of motile algae deep in the

water column is also consistent with the hypothesis thatnutrient availability in the epilimnion is low. Motile phyto-plankton stratify in response to light, nutrient availability,and temperature. The dinoflagellate Ceratium sp. congre-gates in layers and avoids anoxia in the hypolimnion andhigh light intensities at the surface. It also migrates down-ward when nutrients are depleted in upper layers (9, 10). Thealgae in the two study lakes were concentrated in thin stratajust above the hypolimnia, even though light intensity at thisdepth was suboptimal for photosynthesis. Algae may there-fore be forced to position themselves close to the sedimentsbecause of severe nutrient limitation elsewhere in the watercolumn.

Release of nutrients such as phosphate from sedimentsoccurs most rapidly under anoxic conditions (35). Severalacid mine lakes in Indiana were found to have permanentlyanoxic sediments (R. W. Smith, M.S. thesis, Indiana Uni-versity, Bloomington, 1971). This was attributed to chemicaldensity gradients that resulted from the precipitation andaccumulation of compounds in bottom waters of higher pH.The density gradient prevented homogeneous mixing of thewater columns in spring and fall. Although the hypolimnionof Reservoir 29 contained higher concentrations of dissolvedsubstances than the surface waters did, the anaerobic regionof higher pH was disrupted in each of the study years byturbulent mixing in the spring and fall. In contrast, Lake Bhad an irregular bottom surface, with holes 8 to 9 m deep.High concentrations of reduced iron were measured in theseholes throughout the year, even when the upper water wasisothermal.

Levels of benthic algal biomass usually increase whenwhole lakes or enclosures are acidified (7, 11, 33). Becausebenthic organisms accumulate in areas where nutrients aremost available in nonacid systems, Dillon et al. (6) haveinferred that the sediments may serve this purpose in acidlakes. For planktonic algae in Reservoir 29, the high H2Sconcentration in the hypolimnion forms a lethal barrierpreventing further downward migration.The pH within the permanently anoxic regions of Lake B

was close to neutrality. Although it is the isolation of thisregion from the remainder of the water column whichpreserves its high pH, microbial activity may have catalyzedthe pH increase. Anaerobic microbial processes, such as thereduction of sulfate to hydrogen sulfide, do reduce acidityand increase pH (16, 17). After Reservoir 29 sedimentsbecame anoxic, increases in H2S concentration and pH weremeasured. Gyure (Ph.D. thesis) has shown that the sulfatereduction is biologically mediated in this system. Althoughthe pH rose only 1 unit during the summer, acidity titrationsillustrated that the hypolimnetic waters were buffered.Therefore, alkalinity generation was significant in the sedi-ments. In terms of biological restoration of Reservoir 29, twosignificant problems are (i) increasing the rate of a processcatalyzed by chemoheterotrophic bacteria in an oligotrophicsystem and (ii) ensuring that reduced sulfide is not reoxid-ized by exposure to oxygen, so that alkalinity is permanentlygenerated.

Measurements of glucose metabolism by heterotrophic

bacteria suggest that the sediment/water interface in Reser-voir 29 is an important site of activity. This reflects thevertical distribution of particulate organic matter: most ofthe algal biomass was located below the thermocline. Therelative rates of [14C]glucose metabolism will be affected bythe size of the glucose pool at different depths. The largestdilution of radioactive glucose would be expected at the lakebottom, where the amount of particulate organic matter ishighest. However, despite the potential effect of pool dilu-tion, ['4C]glucose metabolism was greatest in this stratum.In contrast, when thymidine incorporation was used as ameasure of heterotrophic activity, activities were similar inthe epilimnion and at the sediment/water interface. In neu-tral-pH systems, heterotrophy measurements also dependon the substrate used (26). However, the bacterial produc-tion estimates obtained with [3H]thymidine for Reservoir 29were similar in magnitude to those for a mesotrophic Indianalake of pH 8 (23). Therefore, low pH itself does not preventheterotrophic activity in highly acid environments. This hasalso been shown for cellulose decomposition in the watercolumn of an acidic lake (13).

In summary, these acid mine lakes have unique character-istics that distinguish them from other neutral or acid fresh-water systems. Low allochthonous carbon and nutrient inputcharacterize Reservoir 29 as oligotrophic, although algalbiomass is higher than would be expected for this trophicstate. In both lakes, algae or photosynthetic bacteria areconcentrated in a thin layer at the anoxic hypolimnia, wherelight intensity is low, and therefore the organisms are notmajor contributors to overall primary production. Specificphotosynthetic rates are low, and this suggests that avail-ability of nitrogen, phosphorus, or CO2 may be more signif-icant limiting factors than the direct effect of low pH.Bacterial and chemical processes in the sediment allowcarbon and nutrient recycling, although high H2S concentra-tion in the hypolimnion of Reservoir 29 can act as a lowerbarrier to vertical migration of motile algae to the nutrient-rich sediment/water interface.

ACKNOWLEDGMENTS

We thank Jed Weber, Kyle Carr, Lenore Ignasiak, John Miner,Charles White, and Robert Philips for technical assistance.

This research was supported in part by the Purdue Water Re-sources Center (project G905-06).

LITERATURE CITED

1. American Public Health Association. 1975. Standard methods forthe examination of water and wastewater, 14th ed. AmericanPublic Health Association, Washington, D.C.

2. Brock, T. D. 1978. Thermophilic microorganisms and life at hightemperatures. Springer-Verlag, New York.

3. Burnison, B. K. 1980. Modified dimethyl sulfoxide (DMSO)extraction for chlorophyll analysis of phytoplankton. Can. J.Fish. Aquat. Sci. 37:729-733.

4. Campbell, R. S., and 0. T. Lind. 1969. Water quality and agingof strip-mine lakes. J. Water Pollut. Control Fed. 41:1943-1955.

5. Cline, J. 1969. Spectrophotometric determination of hydrogensulfide in natural waters. Limnol. Oceanogr. 24:454-459.

6. Dillon, P. J., N. D. Yan, and H. H. Harvey. 1983. Acidicdeposition: effects on aquatic ecosystems. Crit. Rev. Environ.Control 13:167-194.

7. Hall, R. J., G. E. Likens, S. B. Fiance, and G. R. Hendrey. 1980.Experimental acidification of a stream in the Hubbard BrookExperimental Forest, New Hampshire. Ecology 61:976-981.

8. Hargreaves, J. W., E. J. H. Lloyd, and B. A. Whitton. 1975.Chemistry and vegetation of highly acidic streams. Freshwater

VOL. 53, 1987

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

Biol. 5:563-576.9. Heaney, S. I., and R. W. Eppley. 1981. Light, temperature, and

nitrogen as interacting factors affecting diel vertical migrationsof dinoflagellates in culture. J. Plankton Res. 3:331-344.

10. Heaney, S. I., and J. F. Tailing. 1980. Dynamic aspects ofdinoflagellate distribution patterns in a small productive lake. J.Ecol. 68:75-94.

11. Hendrey, G. R. 1976. Effects of pH on the growth of periphyticalgae in artificial stream channels. SNSF IR 25/72. Sur NedborsVirkning pa Skog og Fisk Project, Oslo.

12. Hobbie, J. E., and C. C. Crawford. 1969. Respiration correc-tions for bacterial uptake of dissolved organic compounds.Limnol. Oceanogr. 14:528-532.

13. Hoeniger, J. F. M. 1986. Decomposition studies in two centralOntario lakes having superficial pHs of 4.6 and 6.6. Appl.Environ. Microbiol. 52:489-497.

14. Ingvorsen, K., and B. B. Jorgensen. 1979. Combined measure-ment of oxygen and sulfide in water samples. Limnol. Ocean-ogr. 24:390-393.

15. Joseph, J. M. 1953. Microbiological study of acid mine waters:preliminary report. Ohio J. Sci. 53:123-137.

16. Kelly, C. A., J. W. M. Rudd, R. B. Cook, and D. W. Schindler.1982. The potential importance of bacterial processes in regu-lating rate of lake acidification. Limnol. Oceanogr. 27:868-882.

17. Kilham, P. 1982. Acid precipitation: its role in the alkalization ofa lake in Michigan. Limnol. Oceanogr. 27:856-867.

18. King, D. L., J. J. Simmler, C. S. Decker, and C. W. Ogg. 1974.Acid strip mine lake recovery. J. Water Pollut. Control Fed. 46:2301-2315.

19. Konopka, A. 1983. The effect of nutrient limitation and itsinteraction with light upon the products of photosynthesis inMerismopedia tenuissima (Cyanophyceae). J. Phycol. 19:403-409.

20. Konopka, A. 1983. Epilimnetic and metalimnetic primary pro-duction in an Indiana hardwater lake. Can. J. Fish. Aquat. Sci.40:792-798.

21. Kwiatowski, R. E., and J. C. Roff. 1976. Effects of acidity on thephytoplankton and primary productivity of selected northernOntario lakes. Can. J. Bot. 54:2546-2561.

22. Likens, G. E. 1975. Primary production of inland aquatic eco-systems, p. 185-202, H. Lieth and R. H. Whittaker (ed.), Theprimary productivity of the biosphere. Springer-Verlag, NewYork.

23. Lovell, C. R., and A. Konopka. 1985. Primary and bacterialproduction in two dimictic Indiana lakes. Appl. Environ. Mi-

crobiol. 49:485-491.24. McConathy, J. R., and J. B. Stahl. 1982. Rotifera in the plankton

and among filamentous algal clumps in 16 acid strip-mine lakes.Trans. Ill. State Acad. Sci. 75:85-90.

25. Muller, P. 1980. Effects of artificial acidification on the growthof periphyton. Can. J. Fish. Aquat. Sci. 37:355-363.

26. Novitsky, J. A. 1983. Microbial activity at the sediment-waterinterface in Halifax Harbor, Nova Scotia. Appl. Environ. Mi-crobiol. 45:1753-1766.

27. Overrein, L. N., H. M. Scip, and A. Tollan. 1980. Acid precip-itation-effects on forest and fish. Final Report of the SNSF-Project 1972 to 1980. Sur Nedbors Virkning pa Skog og FiskProject, Oslo.

28. Riemann, B., J. Fuhrman, and F. Azam. 1982. Bacterial second-ary production in freshwater measured by [3H]-thymidine incor-poration method. Microb. Ecol. 8:101-114.

29. Schindler, D. W., K. H. Mills, D. F. Malley, D. L. Findlay, J. A.Shearer, I. J. Davies, M. A. Turner, G. A. Linsey, and D. R.Cruikshank. 1985. Long-term ecosystem stress: the effects ofyears of experimental acidification on a small lake. Science228:1395-1401.

30. Schofield, C. L. 1976. Acid precipitation: effects on fish. Ambio5:228-230.

31. Shellito, G. A., and J. DeCosta. 1981. Primary production in aeutrophic acid lake. Water Air Soil Pollut. 16:415-431.

32. Singer, P. 1970. Oxygenation of ferrous iron. FWQA report no.14010-06/69. U.S. Department of the Interior, Washington, D.C.

33. Stokes, P. M. 1981. Benthic algal communities in acid lakes, p.119-138. In R. Singer (ed.), Effects of acid precipitation onbenthos. North American Benthic Society, Canterbury Press,New York.

34. Strickland, J. D., and T. R. Parsons. 1972. A practical handbookof seawater analysis, 2nd ed., Bulletin 167. Bull. Fish. Res.Board Can.

35. Stumm, W., and J. J. Morgan. 1970. Aquatic chemistry. JohnWiley & Sons, Inc., New York.

36. Tabatabai, M. A. 1974. Determination of sulfate in watersamples. Sulfur Inst. J. 10:11-13.

37. Temple, K. L., and E. W. Delcamps. 1952. Autotrophic bacteriaand the formation of acid mine drainage: preliminary report.AppI. Microbiol. 1:255-268.

38. Tuttle, J. H., P. R. Dugan, C. B. MacMillan, and C. I. Randles.1969. Microbial dissimilatory sulfur cycle in acid mine water. J.Bacteriol. 97:594-602.

39. Wetzel, R. G. 1975. Limnology, p. 331-354. The W. B. SaundersCo., Philadelphia.

2076 GYURE ET AL.

on August 27, 2020 by guest

http://aem.asm

.org/D

ownloaded from