Abstract

Th e Kimble Creek abandoned coal mine site, located on Wayne National Forest 

in southeastern Ohio, is among several abandoned coal mine sites that have been 

responsible for the acid mine drainage (AMD) polluting ground and surface water. 

Materials released by AMD include iron, aluminum, manganese, other hazardous 

substances, and acidity that are harmful to aquatic life. We tested at the Kimble Creek 

site the Pyrolusite Process®, a microbiological water treatment system that employs 

metal-oxidizing bacteria adsorbed on limestone rocks. Th e pilot treatment plant 

constructed was inoculated with a mixture (fi ve strains) of metal-oxidizing bacteria. 

Water was examined at regular intervals for pH, concentrations of metals (Fe, Al, 

Mn, Mg, Zn, Ca), dissolved oxygen, temperature, conductivity, hardiness, sulfate 

and chloride contents, acidity, alkalinity, and dissolved solids. Results showed that 

the Prylosite Process consistently increased the pH of effl uent water to above-neutral 

levels. Th e iron and aluminum contents, which were as high as 72 mg/L and 50 mg/L, 

respectively, were below detectable levels in the effl uent water. Scanning electron 

microscope (SEM) and DNA analyses of rock samples showed growth and density 

increases of inoculated bacteria. Th e sludge contained iron (predominantly Fe+3), 

aluminum, and small amounts of zinc, magnesium, and manganese. Based on the 

performance of the pilot system, a full-fl edged system was constructed at the Kimble 

Creek site which is functioning as expected based on preliminary tests.

Pyrolusite Process® to Remove 

Acid Mine Drainage Contaminants 

from Kimble Creek in Ohio: 

A Pilot Study

Shiv Hiremath, Kirsten Lehtoma,

Mike Nicklow, and Gary Willison

United States Department of Agriculture

Forest 

Service

Research Note

NRS-194

Northern 

Research Station November 2013

Th e use of trade, fi rm, or corporation names in this publication is for the information and 

convenience of the reader. Such use does not constitute an offi cial endorsement or approval 

by the U.S. Department of Agriculture or the Forest Service of any product or service to the 

exclusion of others that may be suitable.
2

INTRODUCTION

Abandoned coal mines produce acid mine drainage (AMD) that pollutes ground water 

supplies. Th e materials in AMD vary by site, but typically include heavy metals (such as 

iron, aluminum, and manganese), other hazardous substances, and acidic water that harms 

aquatic life. Improved economical methods are needed to remove these substances from 

water that otherwise could have detrimental eff ects on forest health and related wildlife.

Th e Kimble Creek abandoned coal mine is located in Lawrence County, within the 

Wayne National Forest in southeastern Ohio. Th e location, a moderately steep, forested, 

north-south trending hill, has three mining-related benches crossing the hillside. Th ere 

are three known portals and another suspected portal along the lowermost of the three 

benches. All three portals drain into Kimble Creek, which ultimately fl ows into the Ohio 

River. Coal waste, known as gob piles, is located near or downhill from these portals 

and also contributes to the AMD. Th e AMD emerging at this site crosses the bench and 

fl ows downhill through gob, coalescing into a single fl ow path at the toe of the gob. 

Flow measurements have shown that water volume increases from the portal to the toe 

of the gob, indicating that subsurface fl ow through the gob is augmenting surface fl ow. 

Maximum fl ow measured at the toe was at 15 gallons per minute.

Th e AMD at the site became evident after a 1995 storm when orange-tinted water was 

observed in Kimble Creek; farther downstream, a fi sh kill occurred in Pine Creek. Th e 

Forest Service and other Federal and State agencies tested water quality and concluded 

that it was related to AMD and included pollutants, or contaminants, as defi ned by 

Comprehensive Environmental Response, Compensation and Liability Act (also known as 

the Superfund law) section 101(14), low pH surface water discharge from an abandoned 

coal mine entrance, and low pH seepage from coal mine gob piles. Th e Pine Creek water 

samples also exhibited iron hydroxide precipitate, abnormal surface water chemistry, 

and lack of normal stream fauna. Investigations also found that surface water leaving the 

abandoned mine site contained concentrations of metals and net acidity known to be 

detrimental to aquatic life.

A preliminary assessment/site inspection (PA/SI) and an engineering evaluation/cost 

analysis (EE/CA) concluded that a 0.4-acre area within the underground mine is partially 

fi lled with approximately 168,000 gallons of acidic water. Th e water exiting the mine as 

surface fl ow through one or more portals is responsible for the observed AMD. Th e EE/

CA surveyed 14 removal action options and investigated three options in detail: 1) mine 

dewatering with expanded fl ow diversion; 2) mine dewatering with limited fl ow diversion 

and bioaugmentation; and 3) microbiological treatment system for pollution removal, 

known as the Pyrolusite Process® (Vail and Riley 1995, 1997, 2000; Allegheny Mineral 

Abatement, no date).

In the Pyrolusite Process®, AMD-aff ected water passes through a limestone ‘bed’ on which 

suitable naturally occurring metal-oxidizing bacteria grow. Th is method described and 

patented by Vail and Riley (1995, 1997, 2000; Allegheny Mineral Abatement) appears 

Th e Authors

SHIV HIREMATH is a 

research molecular biologist 

with the U.S. Forest Service, 

Northern Research Station, 

Delaware, OH 43015.

KIRSTEN LEHTOMA is a 

biological technician with the 

U.S. Forest Service, Northern 

Research Station, Delaware, 

OH 43015.

MIKE NICKLOW is an 

environmental engineer 

with the U.S. Forest Service, 

Wayne National Fore st, 

Nelsonville, OH 45764.

GARY WILLISON is the 

watershed wildlife botany fi re 

timber and engineering group 

leader with the U.S. Forest 

Service, Wayne National 

Forest, Nelsonville, OH 

45764.
3

to be very eff ective compared to several passive systems described for removal of iron, 

aluminum, and acidity from AMD (Faulkner and Skousen, 1993, 1994; Hedin et al. 

1994a, b; Rose and Dietz 2002; Watzlaf et al. 2000; Ziemkiewicz et al. 1997). Similar 

methods of using microbial mitigation reactors have also been reported (Bechard et al. 

1993, 1994; Cairns et al. 1988; Mills 1985).

Because of potential problems associated with options 1 and 2 that might have impeded 

success of the remediation project, we decided to investigate the feasibility of the third 

option through a pilot project before a full-scale system is employed for bioremediation of 

water at the Kimble Creek site.

In this paper we describe the results of the pilot study.

MATERIALS AND METHODS

Th e Pyrolusite Process® consists of a limestone “bed” inoculated with selected bacteria. 

Th ese bacteria sustain growth in an oxygenated environment and are nonpathogenic to 

plants and animals. As the bacteria grow on rocks, they etch away the limestone, releasing 

calcium carbonate to neutralize the acidity of the AMD and simultaneously oxidize metals 

to remove them from water (Vail and Riley 1995, 1997, 2000).

Removal of manganese under these conditions is achieved at a pH 6 to 7, while 

standard chemical treatment would require the pH to be raised to 9.5. Iron, which is 

a major component of AMD and normally precipitates out as a voluminous hydroxide 

(yellowboy) at pH 4, is removed as a dense, crystalline, low-volume oxide. Th e removal 

of iron as oxide prevents the system from clogging and keeps water fl owing unobstructed. 

Th is system also removes aluminum abiotically when the pH increases.

For systems such as these, limestone with the highest calcium content is preferred. Th e 

advantages of using limestone include low cost, ease of use, and formation of a dense, 

easily handled sludge. Th e disadvantages include slow reaction time, loss in effi ciency of 

the system because of coating of the limestone particles with iron precipitates, diffi culty 

in treating acid mine drainage with a very high ferrous-ferric ratio, and ineff ectiveness 

in manganese removal. However, the utilization of microbes mitigate most of these 

disadvantages.

Th e underground treatment plant designed by Robert Kerber of the Wayne National 

Forest is shown in Figure 1. It had three waterproof compartments roughly 50 ft wide, 15 

ft long, and about 5 ft deep. Each compartment contained a bed of limestone.

Th e infl uent water (a part of the total fl ow) entered from bottom of the fi rst compartment, 

fl owed into the second compartment from the top, entered the third compartment from 

bottom, and fi nally exited the plant from the top.
4

Th e retention time was about 1,500 cubic ft limestone/gallon/min of fl ow and the plant 

was set for an average fl ow rate of 2 to 4 gallon per minute and a maximum of 15 gallons 

per minute. Th e plant was equipped with ports for obtaining water and rock samples 

(black rectanges 1-6, Fig. 1), as well as ports for inoculating the limestone bed with 

bacteria (In1-6, Fig. 1).

Design was completed in March 2003, construction was completed in May 2003, fi rst 

inoculation was done in May 2003, and initial samples were collected in June 2003.

Signifi cant changes in the plant design were made after the plant was in operation for 6 to 

12 months. In the original design, water from the hillside portals was pooled at the top of 

the hill, fl owed through an underground pipe, and entered the fi rst compartment from the 

bottom. However, the oxygen content of the water was too low, which probably caused 

reduced oxidation of metals leading to a clogged system. To correct this, a manifold was 

added near the plant entrance to aerate the infl uent water. In the revised system, water 

fl owed through a manifold and entered the fi rst compartment from the top.

Suboptimal bacterial growth on the limestone surfaces led to another change. Th is was 

traced to an inadequate carbon source required by the bacteria.

Th erefore, an upstream holding cell covered with vegetation was added to provide more 

nutrients essential for the bacteria. Th is design was not initially considered since the site 

was thickly vegetated. However, after poor performance of the system in early periods, it 

was added to improve growth of bacteria.

Figure 1.—Aerial view of pilot treatment plant layout and sample collection locations. The treatment plant consisted of 3 

water-proof cells, influent water entering cell 1 and exiting from cell 3. Black rectangular icons (numbered 1 to 6) represent 

ports from where water and rock samples were obtained for analyses. Locations 1 and 6 represent the influent and 

effluent water, respectively. In1-6 (green) indicate locations of ports for inoculating the system with bacteria. The system 

was designed such that the sediment could be flushed out from the bottom of the plant to obtain the sludge.

1 

2 

3 

4 

5 

6 

In1 

In4 

In5 

In6 

In2 

In3 

Sludge 

(Influent) 

(Effluent) 
5

Inoculation

Th e limestone bed was inoculated with bacteria grown on rock surfaces as well as liquid 

medium through inoculation ports by the Allegheny Mineral Abatement, Inc. Th e liquid 

medium consisted of a mixture of fi ve uncharacterized metal oxidizing bacteria described by 

Vail and Riley (2000).

Monitoring

Samples of infl uent water, effl uent water, and water from internal points in the plant were 

collected monthly. A portion of the water sample was acidifi ed with nitric acid immediately 

after collection for measuring certain characteristics. To measure the Fe+2 content, a portion 

of the sample was preserved in air-tight sample containers to limit spontaneous oxidation. 

Most chemical analyses were performed by the Ohio Environmental Protection Agency at 

the agency’s Reynoldsburg, Ohio, laboratory. Th e analysis of iron content (Fe+2 and total) 

was performed by the Aqua Tech Environmental Laboratories, Inc., Marion, Ohio. Water 

samples were analyzed for aluminum, manganese, magnesium, pH, hardness, chloride, 

sulfate, alkalinity, and dissolved oxygen.

Rock samples from the limestone bed (5 rocks from each port) were also collected for 

evaluating presence and growth of bacteria. Bacteria were visualized through a scanning 

electron microscope (SEM) coupled to an x ray machine; x ray analysis used to identify 

elements present on the rock surface.

Bacterial growth on the rocks was estimated by determining the total DNA content. Dry rock 

surfaces were scraped to remove adhering material and subjected to DNA extraction. Th e 

material from each rock was suspended in 1 ml 50 mM Tris-HCl, pH 8.0, containing 10 mM 

EDTA. After disruption for 30 seconds using a Tissuemizer probe attached to a Dremel tool, 

sodium dodecyl sulfate and proteinase K were added to fi nal concentrations of 0.5 percent 

and 100 μg/ml, respectively. Th e mixture was incubated at 37 °C for 1 hour. Sodium chloride 

(0.7 M fi nal) and hexadecyltrimethylammonium bromide (CTAB) (1% fi nal) were added and 

incubated at 65  ̊C for 10 minutes. Th e resulting mixture was extracted with an equal volume 

of a mixture of chloroform:isoamyl alcohol (24:1). Th e aqueous portion containing DNA 

was collected after brief centrifugation and the DNA concentration was determined using a 

Pharmacia (Basking Ridge, NJ) GeneQuant spectrophotometer.

Sludge Analysis

Th e plant bed was fl ushed and all the sediment removed after 8 months. Th e sludge was 

examined for the presence of metals and hazardous materials. Also, analysis was performed 

to measure the forms of iron (Fe+2 or Fe+3).

RESULTS AND DISCUSSION

We looked at three main parameters to assess usefulness of the Pyrolusite Process® in 

improving the water quality at the Kimble Creek site: 1) pH and metal content of infl uent 

and effl uent water; 2) growth of bacteria and metal deposition on rock surfaces; and 3) the 
6

sludge. Sludge was analyzed to determine whether bacteria were oxidizing iron hydroxides 

to convert into ferric oxides, the quantity of sludge generated, and to test whether the 

sludge is nontoxic/nonhazardous for disposal according to U.S. Environmental Protection 

Agency standards.

Th e original plan was to monitor the system for a 12- to 24-month period after it was 

in operation. However, in the fi rst year the system did not function well because of the 

poor design as previously described. After modifi cations, the pilot study was extended for 

almost 3 years (until 2007).

One of the major modifi cations was the addition of a manifold to increase oxygen content 

of the water. It was essential because the low dissolved oxygen content in the water was 

not enough for the bacteria to carry out oxidation. Th is resulted in the accumulation 

of voluminous ferrous sulfate which was responsible for the poor fl ow and signifi cant 

reduction in removal of iron (see Table 1). After the manifold was added, there was 

signifi cant increase in the dissolved oxygen level which resulted in unobstructed fl ow 

through the system and removal of iron. Th e second major modifi cation was that of 

adding an upstream holding cell covered with vegetation. Th is was constructed for dual 

purposes: to increase AMD collection system and bacterial nutrients, and to provide an 

oxygenated environment. Th ese modifi cations resulted in signifi cant improvement of the 

system’s performance. Although the pilot study was extended for 3 years, we present in 

this report results of the initial period: 2004 to 2005.

Sample Collection

Fig. 1 shows the diagram of the treatment plant and ports from which samples were 

collected every month. Although the main goal was to test the quality of infl uent and 

effl uent water, samples from within diff erent compartments were also tested to get an 

idea about the functioning of the plant. Locations 1 and 6 represent the infl uent and 

effl uent water, respectively. We arbitrarily chose locations 3 and 5 for routine analyses 

to understand functioning within diff erent compartments, and later added location 4 

to this list. Rock samples were collected from all six locations. Since there were some 

questions about whether adequate oxygen was available inside the plant for the growth 

and functioning of the bacteria, the dissolved oxygen (and water temperature) was also 

measured at all locations.

Water Analyses

Th e Pyrolusite Process® is designed to use bacteria to oxidize ferrous (Fe+2) hydroxides 

to the crystalline ferric (Fe+3) oxide form. To test whether oxidation was occurring at the 

rock surface, we analyzed water samples for Fe+2 as well as total iron (Fe+2 + Fe+3). Th is 

is a diffi cult task since spontaneous air oxidation can occur during sample collection. 

However, airtight containers were used to limit such oxidation. We also used chemical 

stabilizers to arrest most of such oxidation for measuring other parameters.
7

Analysis showed that while nearly 90 percent of Fe in the infl uent water was in the ferrous 

form (+2), by the time it reached location 3 nearly 75 percent was in the ferric (+3) form 

(data not shown). Samples taken at location 5 had on average less than 1 percent of Fe 

levels present in the infl uent water, and most of it was in the ferric form. Th ese results 

proved that bacteria were indeed oxidizing iron.

Figure 2 shows the amount of iron removed from the water for the period January 2004 

to April 2005; some of the earlier data (July 2003 to April 2004) is summarized in Table 

1. Due to some initial problems (October to December 2003) with the system, iron was 

not completely removed (Table 1). We believe several factors were probably responsible. 

Bacteria had been just introduced and may not have had enough time to establish on the 

rocks. Also, during these months the amount of iron in the infl ow water was unusually 

high compared to what had been observed earlier. However, the major cause was traced 

to two factors: inadequate dissolved oxygen essential for bacteria in the oxidation process, 

and inadequate nutrients necessary for sustained growth of bacteria. Th is was based on 

Figure 2.—Iron analysis for the period January 2004-April 2005. Although we monitored samples from 

all port locations, the figure shows data for the influent (yellow) and effluent (blue) water samples. Iron 

content is expressed as mg/L.

0

20

40

60

80

100

120

140

160

inflow Fe

out Fe

Table 1.—Iron removal data for Kimble Creek

Iron Content pH

Month Influent Effluent % Removed Influent Effluent

July 59.8 mg/L <0.05 mg/L 100 4.0 7.2

August 53.0 26.8 49 3.27 6.79

September 46.5 5.83 87 3.49 7.38

October 72.1 29.0 60 3.31 6.85

November 35.5 19.0 46 3.77 6.92

December 62.1 14.0 77 3.63 7.15

January 31.5 <0.05 100 4.94 7.49

February 19.5 <0.05 100 4 .81 7.62

March 15.2 <0.05 100 4.97 7.48

April 36.6 <0.05 100 4.91 7.30
8

actual measurement of dissolved oxygen in water samples and bacterial growth on the 

rocks. After the addition of the manifold and inserting an upstream holding cell, the plant 

was able to remove almost 100 percent of iron present in infl uent water.

Th e pH data followed a similar profi le to iron in water samples. Th e data in Fig. 3 and 

Table 1 showing the consistently above-neutral pH of the effl uent water indicated that the 

system was functioning effi ciently as expected.

Data also indicate that there are seasonal changes in the quality of the infl uent water. 

In cold months, the pH was typically higher and amount of iron was low. It is possible 

that during these months, there is less solubilization of iron at the site of origination 

and, therefore, less iron in the AMD. Th is is supported by observed increases in the iron 

content as soon as the environmental temperature increased (April onwards). In any case, 

the system appeared to function well throughout the seasonal cycle after the fi rst year.

We also measured dissolved oxygen, temperature, conductivity, concentrations of other 

metals (Al, Mn, Mg, Mn, Zn, Ca), hardness, sulfate and chloride content, acidity, 

alkalinity, and dissolved solids. We do not present all the data in this report. However, 

aluminum was the second most common contaminant observed in the AMD at the 

Kimble Creek site. Th e levels, which were as high as 50 to 100 mg/L during some 

months, were reduced to almost undetectable levels in the effl uent samples. Another major 

pollutant, manganese, which was as high 1 to 1.5 mg/L , was also completely removed 

by the system. In summary, the effl uent water contained no AMD contaminants at levels 

considered harmful.

Data also indicates that the acidity of the infl uent water was almost completely removed 

by the Pyrolusite Process® (Fig. 4). Th e effl uent water consistently had a pH near or above 

neutral range.

Figure 3.—pH measurements of influent (purple) and effluent (red) water samples.

0

1

2

3

4

5

6

7

8

9

inflow pH

outflow pH
9

Rock Analyses

We assessed the presence and density of bacteria through a scanning electron microscope 

(SEM) and DNA measurements. Th e SEM analysis showed presence of bacteria on rock 

surfaces (Fig. 5). Monthly monitoring suggested that bacterial population on the rocks 

steadily increased and showed no major diff erences through seasonal changes.

We also empirically measured bacterial density by determining total DNA mass on the 

rock surface. Th is provided a comparative measurement to determine relative growth 

of bacteria. Results (data not shown) showed that there was a steady increase in DNA 

mass on the rock surfaces post-inoculation. Th is would be expected if inoculated bacteria 

colonized rocks and kept growing over time. However, since these were total DNA 

measurements, it was not possible to confi rm whether the DNA measured is from the 

inoculated 

bacteria or from 

other bacteria or 

organisms that 

colonized rock 

surfaces during the 

same period.

Figure 4.—Acidity (mg/L) and alkalinity (mg/L) of influent (dark blue) and effluent samples (light blue).

Figure 5.—Bacteria growth on rock surface, shown at 1,000× magnification 

in this image.

0

50

100

150

200

250

300

350

400

450

acid in

alk out
10

We also subjected rock surfaces to elemental analysis using SEM coupled to a x ray 

machine (Fig. 6). Such analysis shows presence of elements including metals present 

on the rock surface. Since the probe analyzed only a tiny spot on the rock surface each 

time, the data gave only a general idea about the contents on the rock and could not be 

extrapolated to the entire rock surface. For each rock piece, we measured three or four 

suitable locations. Th e analysis gave a rough idea about metal deposition and a clue to 

whether extensive armorization was occurring on the rock surface. Th e latter phenomenon 

leads to clogging and slowing/stoppage of water fl ow. Fe on rocks in location 5 was 

signifi cantly lower than in location 2 (Fig. 6). Th is is to be expected since most of the iron 

precipitated as soon as the pH increased in compartment 1, thereby leaving little iron 

moving into subsequent compartments of the treatment plant.

Sludge analysis

After 12 months of operation, the system was fl ushed and the sludge was analyzed. Many 

pollutant metals from AMD were present in the sludge, but the highest levels were of 

Fe, Al, and Mn. Th e solid part of the sludge was estimated to contain 178 g Fe per kg of 

sludge, 85.7 g Al per kg of sludge, 17.5 g Ca per kg of sludge, 0.85 g Mn per kg of sludge, 

1.94 g of Mg per kilogram of sludge, and 0.59 g Zn per kilogram of sludge.

Since we wanted to learn more detail about the oxidation process in the Pyrolusite 

Process®, we tried to determine the nature of Fe (ferrous [Fe+2] and ferric [Fe+3] 

forms) in the sludge sediment. Although it is very diffi cult to stop air oxidation of Fe+2

during collection, it was minimized by using stabilizers. It is not easy to accurately 

determine relative amounts of Fe+2 in solid samples, however, a colorimetric method 

provided a means to approximate the percentage of Fe+2 and Fe+3 in the sample. Such 

Figure 6.—Elemental analysis of rocks using a scanning electron microscope equipped with a x ray machine. 

At location 2, iron could be seen whereas by the time water reached location 5, it was hardly detectable. Similar 

results were observed with aluminum depositions.

Location 2 Location 5

Energy (keV) Energy (keV)
11

analyses showed that 90 percent of iron was Fe+3and only about 10 percent Fe+2. 

Finally, the sludge sample was subjected to a toxicity characteristic leaching procedure 

(TCLP) digestion/analysis to test for hazardous material leaching. Hazardous material 

amounts were below detectable levels (data not shown) indicating that the material was 

nonhazardous as per U.S. Environmental Protection Agency regulations and could be 

disposed as solid waste.

CONCLUSIONS

Th e Pyrolusite Process® performed very eff ectively in removing the pollutants from the 

AMD at the Kimble Creek coal mine site. Results in 2006 to 2007 were similar to what 

was seen in the latter part of 2005 with minor seasonal changes. Results from this pilot 

study showed that this system could be employed to improve quality of water fl owing into 

the Kimble Creek. A large-scale system was constructed in 2008 and water from all portals 

was directed through the system. Th e large-scale plant has been functioning well and has 

been eff ective in increasing the pH and removing all the pollutants contributed by the 

AMD.

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12

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FL. Lexington, KY: American Society of Mining and Reclamation: 262-274.

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Hiremath, Shiv; Lehtoma, Kirsten; Nicklow, Mike; Willison, Gary. 2013. 

Pyrolusite Process® to remove acid mine drainage contaminants 

from Kimble Creek in Ohio: A pilot study. Res. Note NRS-194. 

Newtown Square, PA: U.S. Department of Agriculture, Forest Service, 

Northern Research Station. 12 p.

KEY WORDS: acid mine drainage, AMD, iron removal, microbiological 

treatment system, Pyrolusite Process®
Northern Research Station

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